[go: up one dir, main page]

WO2025207632A1 - Compositions et méthodes pour le traitement de néoplasmes myéloprolifératifs - Google Patents

Compositions et méthodes pour le traitement de néoplasmes myéloprolifératifs

Info

Publication number
WO2025207632A1
WO2025207632A1 PCT/US2025/021343 US2025021343W WO2025207632A1 WO 2025207632 A1 WO2025207632 A1 WO 2025207632A1 US 2025021343 W US2025021343 W US 2025021343W WO 2025207632 A1 WO2025207632 A1 WO 2025207632A1
Authority
WO
WIPO (PCT)
Prior art keywords
pharmaceutical composition
seq
unit
calreticulin
sequence
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/US2025/021343
Other languages
English (en)
Inventor
Sunniva BJØRKLUND
Raquel DEERING
Pierre DILLARD
Anne Laure FLAMAR
Johanna Hansen
Frank Kuhnert
Chia-Yang Lin
Håkan NORELL
Sudesh PAWARIA
O. Gavin Thurston
Michael Van Meter
Christian Winther WOLD
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.)
Regeneron Pharmaceuticals Inc
Original Assignee
Regeneron Pharmaceuticals 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 Regeneron Pharmaceuticals Inc filed Critical Regeneron Pharmaceuticals Inc
Publication of WO2025207632A1 publication Critical patent/WO2025207632A1/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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • 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
    • A61K2039/64—Medicinal preparations containing antigens or antibodies characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Mutations expressed by pre-malignant neoplasms and cancers represent a class of neoantigens for targeting by therapeutic vaccination, such as therapeutic DNA vaccination.
  • Targeting of protein antigens to antigen-presenting cells can improve T- cell and B-cell responses.
  • Recombinant immunoglobulin (Ig) molecules are particularly suited to this purpose.
  • short antigenic epitopes can replace loops between P- strands in the Ig constant domains, while targeted antigen delivery is obtained by equipping the recombinant Ig with variable (V) regions specific for surface molecules on APCs.
  • Calreticulin also called CALR, is a conserved chaperone protein that regulates numerous cellular processes, including protein folding, cell adhesion, and calcium homeostasis. Mutations in calreticulin, particularly frameshift mutations in exon 9, are common driver mutations in myeloproliferative neoplasms (MPN).
  • MPN myeloproliferative neoplasms
  • a pharmaceutical composition comprising a polynucleotide comprising one or more sequences encoding a targeting unit, one or more sequences encoding a dimerization unit, and one or more sequences encoding an antigenic unit, wherein the antigenic unit comprises a sequence encoding at least one epitope derived from calreticulin, and a pharmaceutically acceptable carrier.
  • the at least one epitope derived from calreticulin comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-11.
  • the frameshift mutation in calreticulin comprises an amino acid sequence of SEQ ID NO: 2-3 or 141-158.
  • the frameshift mutation in calreticulin comprises an amino acid sequence of SEQ ID NO: 3.
  • a polynucleotide encoding the frameshift mutation in calreticulin comprises a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 183.
  • a polynucleotide encoding the frameshift mutation in calreticulin comprises SEQ ID NO: 183, or a sequence with 1, 2, 3, 4 or 5 insertions, substitutions or deletions relative thereto.
  • the at least one epitope has a length of 7 to 44 amino acids.
  • the antigenic unit comprises at least two, at least three, at least four, or at least five epitopes from calreticulin.
  • the dimerization unit comprises a hinge region.
  • the hinge region is derived from an immunoglobulin.
  • the dimerization unit further comprises a dimerization domain.
  • the dimerization domain comprises an immunoglobulin constant domain.
  • the immunoglobulin constant domain is a carboxyterminal C domain derived from an IgG.
  • the carboxyterminal C domain is derived from IgG3.
  • a polynucleotide encoding the dimerization unit comprises a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 185.
  • the nucleotide sequence comprises a sequence of SEQ ID NO: 185 or a sequence with 1, 2, 3, 4 or 5 insertions, substitutions or deletions relative thereto.
  • the antigenic unit and the dimerization unit are connected by a linker.
  • the linker comprises an amino acid sequence of SEQ ID NO:98.
  • a polynucleotide encoding the linker comprises a sequence of SEQ ID NO: 187.
  • the targeting unit comprises a moiety that interacts with a surface molecule on an antigen presenting cell.
  • the surface molecule is selected from the group consisting of HLA, CD14, CD40, a chemokine receptor, and a Tolllike receptor.
  • the targeting unit comprises a MIP-lu chemokine that binds to a CCR5 receptor.
  • the MIP-lu chemokine comprises an amino acid sequence of SEQ ID NO: 159.
  • a polynucleotide encoding the MIP-lu chemokine comprises a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 184.
  • the nucleotide sequence comprises a sequence of SEQ ID NO: 184 or a sequence with 1, 2, 3, 4 or 5 insertions, substitutions or deletions relative thereto.
  • the polynucleotide is an RNA or a DNA.
  • the polynucleotide comprises a nucleotide sequence encoding a signal peptide.
  • the signal peptide comprises SEQ ID NO: 163.
  • a polynucleotide encoding the signal peptide comprises a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 186.
  • the nucleotide sequence comprises a sequence of SEQ ID NO: 186 or a sequence with 1, 2, 3, 4 or 5 insertions, substitutions or deletions relative thereto.
  • the polynucleotide comprises, from 5' to 3', one or more sequences encoding the targeting unit, the dimerization unit, and the antigenic unit.
  • the targeting unit encodes a MIP-lu sequence
  • the dimerization unit encodes a hinge exon hl from IgG3, a hinge exon h4 from IgG3, a dimerization unit linker, and human CH3 domain of IgG3, and the antigenic unit encodes an epitope derived from calreticulin comprising SEQ ID NOs:4-l 1.
  • the polynucleotide comprises a sequence encoding a signal peptide of MIP-lu 5' to the targeting unit, and a sequence encoding a linker between the dimerization unit and the antigenic unit.
  • the polynucleotide comprises, from 5' to 3': a targeting unit encoding SEQ ID NO: 159; a dimerization unit encoding SEQ ID NO: 178; and an antigenic unit encoding SEQ ID NO:3.
  • the polynucleotide comprises, from 5' to 3': a signal peptide encoding SEQ ID NO: 163; a targeting unit encoding SEQ ID NO: 159; a dimerization unit encoding SEQ ID NO: 178; a linker encoding SEQ ID NO:98; and an antigenic unit encoding SEQ ID NO:3.
  • the polynucleotide comprises, from 5' to 3': SEQ ID NO: 184; SEQ ID NO: 185; and SEQ ID NO: 183. In some embodiments, the polynucleotide comprises, from 5' to 3': SEQ ID NO: 186; SEQ ID NO: 184; SEQ ID NO: 185; SEQ ID NO: 187; and SEQ ID NO: 183.
  • composition comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 177.
  • an expression vector comprising the polynucleotide of the above aspect.
  • a host cell comprising the polynucleotide or the expression vector of any of the above aspects.
  • polypeptide encoded by the polynucleotide of any of the above aspects or embodiments is provided herein.
  • a dimeric protein comprising two polypeptides of the above aspect.
  • the dimeric protein is a homodimer.
  • the polynucleotide, the polypeptide, or the dimeric protein of the above aspects or embodiments is for use in the manufacture of a medicament for treating a cancer.
  • the cancer is a myeloproliferative neoplasm.
  • the pharmaceutical composition of any one of the above aspects or embodiments is for use in the manufacture of a medicament for treating cancer in a subject.
  • the cancer is a myeloproliferative neoplasm.
  • the cancer is a myeloproliferative neoplasm.
  • a method of eliciting an immune response in a subject in need thereof comprising administering the pharmaceutical composition of any one of the above aspects or embodiments to the subject.
  • the myeloproliferative neoplasm is chronic myeloid leukemia, acute myeloid leukemia, polycythaemia vera, essential thrombocythemia, primary myelofibrosis, chronic eosinophilic leukemia, chronic myelomonocytic leukemia, systemic mastocytosis, idiopathic myelofibrosis, or myeloma.
  • the cancer expresses calreticulin.
  • the calreticulin comprises a frameshift mutation in exon 9.
  • the treatment is therapeutic.
  • the treatment is prophylactic.
  • FIG. 1A shows the results of immunopeptidomic mass spectrometry (IP -MS) for 9- mers of calreticulin and distribution of peptide length. Peptide count is indicated on the y- axis, which indicates from bottom to top: 0, 1000, 2000, 3000, 4000. Peptide length (from 7 to 20 amino acids) is indicated on the x-axis.
  • FIG. IB depicts mass spectra of the endogenous or stable isotope labeled (heavy Spike-in) peptides.
  • SPARPRTSC SEQ ID NO: 5.
  • FIG. 2A is a schematic depiction of an example protocol for antigen-specific memory T cell expansion.
  • PBMC peripheral blood mononuclear cells
  • IFNa interferonalpha
  • GM-CSF Granulocyte-macrophage colony-stimulating factor
  • IL interleukin.
  • FIG. 2B depicts flow cytometry plots for myeloproliferative neoplasm (MPN) patient and healthy donor CD8 + T cells upon stimulation with DMSO, a wild type calreticulin (CALR WT ) peptide pool, a mutant calreticulin comprising the exon 9 frameshift (CALR MUT ) peptide pool, or CEF viral peptide pool (Cytomegalovirus, Epstein-Barr virus, and Influenza, abbreviated CMV, EBV, and Flu respectively).
  • MPN myeloproliferative neoplasm
  • FIG. 2C is a graph depicting a summary of IFNy and TNFa production from MPN patient (right plot) or healthy donor (left plot) CD8+ T cells upon stimulation with DMSO, a CALR MUT peptide pool, a CALR WT peptide pool, or a CEF pool.
  • FIG. 3A is a schematic depicting an example protocol for antigen-specific naive T cell expansion.
  • TUP TNFa, IL-1, IL-6, PGE1 (Prostaglandin El); moDC: monocyte-derived dendritic cell.
  • FIG. 3B is a series of immunofluorescence microscopy images depicting IFNy production using an IFNy ELISpot assay.
  • Naive T cells from Healthy Donor 13 (HD 13) were expanded with DMSO or CALR MUT peptides for 26 days, then stimulated with the indicated peptide pools or DMSO.
  • FIG. 3C is a graph depicting IFNy production measured using an IFNy ELISpot assay.
  • Naive T cells from four donors were expanded with DMSO or CALR MUT peptides for 20 days, then stimulated with a CALR MUT peptide pool, CALR WT peptide pool, or DMSO.
  • HD human donor.
  • 5A depicts a schematic of the construct used to engineer TC-1 cells expressing a HA-Ub-CALR MUT 44-mer construct (left), and flow cytometry validation of intracellular expression in engineered TC-1 cells by HA-tag (HA) detection (right).
  • FIG. 5B provides line graphs depicting in vivo tumor control of the HA-Ub-CALR- 44mer engineered TCI cell line in C57BL/6 mice, vaccinated with Empty VaccibodyTM (negative control) or CALR MUT 44-mer VaccibodyTM, with mice immunized twice by intramuscular injection followed by electroporation at days 7 and 14 post cell implantation.
  • a VaccibodyTM is a multimeric protein containing multiple polypeptides, such as a dimeric protein comprising or consisting of two polypeptides, each a fusion protein comprising a targeting unit, which targets or is capable of targeting antigen-presenting cells, a multimerization unit, such as a dimerization unit, and an antigenic unit.
  • compositions according to the present disclosure are designed for evoking a cell- mediated immune response, e.g., through activation of T cells against the neoantigens as described herein.
  • the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: “A, B, and C”; “A, B, or C”; “A or C”; “A or B”; “B or C”; “A and B”; “B and C”; “A and C”; “A” (A alone); “B” (B alone); and “C” (C alone).
  • the terms “about” and “approximately” refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system.
  • “about” or “approximately” can mean within one or more than one standard deviation per the practice in the art.
  • “about” or “approximately” can mean a range of up to 10% (i.e., ⁇ 10%) or more depending on the limitations of the measurement system.
  • about 5 mg can include any number between 4.5 mg and 5.5 mg.
  • the terms can mean up to an order of magnitude or up to 5-fold of a value.
  • the meaning of “about” or “approximately” should be assumed to be within an acceptable error range for that particular value or composition.
  • the ranges and/or subranges can include the endpoints of the ranges and/or subranges.
  • calreticulin also known as CALR, CALR1, calregulin, SSA, CRT, CRTC, RO, Autoantigen RO, FLJ26680, CClqR, CRP55, ERp60, HACBP, Grp60, Epididymis Secretory Sperm Binding Protein Li 99n, and HEL-S-99n
  • CALR also known as CALR, CALR1, calregulin, SSA, CRT, CRTC, RO, Autoantigen RO, FLJ26680, CClqR, CRP55, ERp60, HACBP, Grp60, Epididymis Secretory Sperm Binding Protein Li 99n, and HEL-S-99n
  • calreticulin also known as CALR, CALR1, calregulin, SSA, CRT, CRTC, RO, Autoantigen RO, FLJ26680, CClqR, CRP55, ERp60, HACBP, Grp60, Epididy
  • wildtype calreticulin refers to a calreticulin polypeptide or protein comprising SEQ ID NO: 1, or a polynucleotide encoding the same.
  • frameshift calreticulin or “calreticulin frameshift mutation” refers to a mutation in the wildtype calreticulin gene (e.g., SEQ ID NO: 1) leading to an alternative open reading frame (ORF).
  • a frameshift mutation in calreticulin occurs in exon 9 (SEQ ID NO: 171).
  • a frameshift mutation may be caused by deletion of one or more nucleotides or by insertion of one or more nucleotides.
  • the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably include humans.
  • nucleic acid refers to polymers of nucleotides and are not limited to any particular length.
  • Nucleic acids include recombinant and chemically- synthesized forms. Nucleic acids can be isolated. Nucleic acids include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids (PNA) and non-naturally occurring nucleotide analogs), and chimeric forms containing DNA and RNA.
  • DNA molecules e.g., cDNA or genomic DNA
  • RNA molecules e.g., mRNA
  • analogs of the DNA or RNA generated using nucleotide analogs e.g., peptide nucleic acids (PNA) and non-naturally occurring nucleotide analogs
  • PNA peptide nucleic acids
  • Nucleic acids can be single-stranded or double-stranded. Nucleic acids comprise polymers of nucleotides, where the nucleotides may include natural or non-natural bases, and/or sugars. Nucleic acids comprise naturally-occurring internucleosidic linkages, for example phosphodiester linkages. Nucleic acids can lack a phosphate group. Nucleic acids can comprise non-natural internucleoside linkages, including phosphorothioate, phosphorothiolate, and/or peptide nucleic acid (PNA) linkages. In some embodiments, nucleic acids comprise one type of polynucleotides or a mixture of two or more different types of polynucleotides.
  • the term “effective amount” or “therapeutically effective amount” refers to the amount of a compound (e.g., a nucleic acid or protein of the present disclosure) sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular administration route.
  • treating includes any effect, e.g., lessening, reducing, modulating, ameliorating or eliminating, that results in the improvement of the condition, disease, disorder, and the like, or ameliorating a symptom thereof.
  • treatment may be preventative (z.e., prophylactic, to reduce the likelihood of the condition, disease, disorder, and the like).
  • composition refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.
  • the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives.
  • stabilizers and adjuvants see e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA [1975],
  • the term “pharmaceutically acceptable salt” refers to salts of such compounds that are appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977).
  • compositions specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the previous definition of the variable controls.
  • An antigenic unit of the polynucleotides of the present disclosure comprises one or more neoepitopes.
  • neoepitopes e.g., an epitope derived from calreticulin
  • MHC major histocompatibility complex
  • MHC I is found on the cell surface of all nucleated cells in the body.
  • One function of MHC I is to display peptides of non-self proteins from within cells to cytotoxic T cells.
  • the MHC I complex-peptide complex is inserted into the plasma membrane of the cell presenting the peptide to the cytotoxic T cells, triggering activation of cytotoxic T cells against the particular MHC -peptide complex.
  • the peptide is positioned in a groove in the MHC I molecule, allowing the peptide to be about 8-10 amino acids long.
  • MHC class II molecules are a family of molecules normally found only on antigen-presenting cells such as dendritic cells, mononuclear phagocytes, some endothelial cells, thymic epithelial cells, and B cells.
  • antigens presented by MHC class II peptides are derived from extracellular proteins. Extracellular proteins are endocytosed, digested in lysosomes, and the resulting antigenic peptides are loaded onto MHC class II molecules and then presented at the cell surface.
  • the antigen-binding groove of MHC class II molecules is open at both ends and is able to present longer peptides than MHC I, generally between 15 and 24 amino acid residues long.
  • Class I MHC molecules are recognized by CD8 and co-receptors on T cells, normally called CD8+ T cells (or CD8+ cells), whereas class II MHC molecules are recognized by CD4 and co-receptors on the T cells, normally called CD4+ T cells (or CD4+ cells).
  • Sequence identity may be determined by any suitable methods known in the art.
  • An example method for determining sequence identity is as follows: a high level of sequence identity indicates likelihood that the first sequence is derived from the second sequence.
  • Amino acid sequence identity requires identical amino acid sequences between two aligned sequences.
  • a candidate sequence sharing 70% amino acid identity with a reference sequence requires that, following alignment, 70% of the amino acids in the candidate sequence are identical to the corresponding amino acids in the reference sequence.
  • Identity may be determined by aid of computer analysis, such as, without limitations, the ClustalW computer alignment program (Higgins D., Thompson J., Gibson T., Thompson J.D., Higgins D.G., Gibson T.J., 1994.
  • the ClustalW algorithm may similarly be used to align nucleotide sequences. Sequence identities may be calculated in a similar way as indicated for amino acid sequences.
  • Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the FASTA sequence alignment software package (Pearson WR, Methods Mol Biol, 2000, 132: 185-219). Align calculates sequence identities based on a global alignment. AlignO does not penalize the gaps in the end of the sequences. When utilizing the ALIGN or AlignO program for comparing amino acid sequences, a BLOSUM50 substitution matrix with gap opening/extension penalties of -12/- 2 may be used.
  • Amino acid sequence variants of the amino acid sequences disclosed herein are contemplated as within the scope of the instant disclosure.
  • Amino acid sequence variants may be prepared by introducing appropriate changes into the nucleotide sequence encoding the composition, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences.
  • the terms substituted/substitution, deleted/deletions and inserted/insertions as used herein in reference to amino acid sequences and sequence identities are well known and clear to the skilled person in the art. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics. For example, deletions, insertions, or substitutions of amino acid residues may produce a silent change and result in a functionally equivalent peptide/polypeptide.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
  • conservative substitutions ie., like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc.
  • non-conservative substitutions i.e., from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine, diaminobutyric acid ornithine, norleucine, ornithine, pyriylalanine, thienylalanine, naphthyl alanine and phenylglycine.
  • Conservative substitutions that may be made are, for example within the groups of basic amino acids (e.g., arginine, lysine and histidine), acidic amino acids (e.g., glutamic acid and aspartic acid), aliphatic amino acids (e.g., alanine, aaline, leucine, isoleucine), polar amino acids (e.g., glutamine, asparagine, serine, threonine), aromatic amino acids (e.g., phenylalanine, tryptophan, tyrosine), hydroxyl amino acids (e.g., serine, threonine), large amino acids (e.g., phenylalanine, tryptophan) and small amino acids (e.g., glycine, alanine).
  • basic amino acids e.g., arginine, lysine and histidine
  • acidic amino acids e.g., glutamic acid and aspartic acid
  • compositions of the present disclosure comprise a polynucleotide encoding a polypeptide comprising three units, i.e. a targeting unit, a dimerization unit, and an antigenic unit. Due to the dimerization unit, the polypeptide forms a dimeric protein called a VaccibodyTM as discussed herein.
  • an antigenic unit according to the disclosure comprises a plurality of tumor neoepitopes.
  • each neoepitope corresponds to at least one mutation identified in a tumor neoantigen.
  • An example of tumor neoepitopes of the disclosure includes epitopes derived from the frameshift region of a calreticulin protein encoded by a sequence comprising a frameshift mutation.
  • the object of the polypeptides is to activate T cells against the neoepitopes.
  • T cells may be diluted in cases where too many neoepitopes or polynucleotides encoding the same are included in the same pharmaceutical composition, and therefore a balance can be struck to provide a pharmaceutical composition with an optimal number of neoepitopes in the antigenic unit.
  • 15 to 50 cancer neoepitopes are included in the polypeptide, or polynucleotide encoding same.
  • the incorporation of 15-50 cancer neoepitopes can elicit an efficient immune response without diluting the T cells when administered to a subject.
  • a neoepitope of the antigenic unit has a length of between 7 to 11 amino acids for MHC class I presentation. In some embodiments, the neoepitope has a length of 9 amino acids for MHC class I presentation.
  • the calreticulin frameshift mutation produces an amino acid sequence comprising a sequence from Table 1 (excluding SEQ ID NO: 1 or SEQ ID NO: 171). In some embodiments, the calreticulin frameshift mutation comprises a sequence of any one of SEQ ID NOs: 2-3 or 141-158.
  • the calreticulin frameshift mutation produces a polypeptide comprising a sequence of SEQ ID NO: 146.
  • the at least one epitope derived from calreticulin comprising a frameshift mutation has a length of about 7 to 44 amino acids from SEQ ID NO: 146.
  • the at least one epitope comprises, consists essentially of, or consists of 7 to 11 amino acids from SEQ ID NO: 146.
  • the at least one epitope comprises, consists essentially of, or consists of 9 amino acids from SEQ ID NO: 146.
  • the at least one epitope comprises, consists essentially of, or consists of 15 amino acids from SEQ ID NO:146.
  • the calreticulin frameshift mutation produces a polypeptide comprising a sequence of SEQ ID NO: 150.
  • the at least one epitope derived from calreticulin comprising a frameshift mutation has a length of about 7 to 44 amino acids and comprises SEQ ID NO: 150 or a portion thereof.
  • the at least one epitope comprises, consists essentially of, or consists of 7 to 11 amino acids from SEQ ID NO: 150.
  • the at least one epitope comprises, consists essentially of, or consists of 9 amino acids from SEQ ID NO: 150.
  • the at least one epitope comprises, consists essentially of, or consists of 15 amino acids from SEQ ID NO: 150.
  • the calreticulin frameshift mutation produces a polypeptide comprising a sequence of SEQ ID NO: 153.
  • the at least one epitope derived from calreticulin comprising a frameshift mutation has a length of about 7 to 44 amino acids from SEQ ID NO: 153.
  • the at least one epitope comprises, consists essentially of, or consists of 7 to 11 amino acids from SEQ ID NO: 153.
  • the at least one epitope comprises, consists essentially of, or consists of 9 amino acids from SEQ ID NO: 153.
  • the at least one epitope comprises, consists essentially of, or consists of 15 amino acids from SEQ ID NO: 153.
  • the calreticulin frameshift mutation produces a polypeptide comprising a sequence of SEQ ID NO: 154.
  • the at least one epitope derived from calreticulin comprising a frameshift mutation has a length of about 7 to 44 amino acids from SEQ ID NO: 154.
  • the at least one epitope comprises, consists essentially of, or consists of 7 to 11 amino acids from SEQ ID NO: 154.
  • the at least one epitope comprises, consists essentially of, or consists of 9 amino acids from SEQ ID NO: 154.
  • the at least one epitope comprises, consists essentially of, or consists of 15 amino acids from SEQ ID NO: 154.
  • the calreticulin frameshift mutation produces a polypeptide comprising a sequence of SEQ ID NO: 155.
  • the at least one epitope derived from calreticulin comprising a frameshift mutation has a length of about 7 to 44 amino acids from SEQ ID NO: 155.
  • the at least one epitope comprises, consists essentially of, or consists of 7 to 11 amino acids from SEQ ID NO: 155.
  • the at least one epitope comprises, consists essentially of, or consists of 9 amino acids from SEQ ID NO: 155.
  • the at least one epitope comprises, consists essentially of, or consists of 15 amino acids from SEQ ID NO:155.
  • the calreticulin frameshift mutation produces a polypeptide comprising a sequence of SEQ ID NO: 156.
  • the at least one epitope derived from calreticulin comprising a frameshift mutation has a length of about 7 to 44 amino acids from SEQ ID NO: 156.
  • the at least one epitope comprises, consists essentially of, or consists of 7 to 11 amino acids from SEQ ID NO: 156.
  • the at least one epitope comprises, consists essentially of, or consists of 9 amino acids from SEQ ID NO: 156.
  • the at least one epitope comprises, consists essentially of, or consists of 15 amino acids from SEQ ID NO: 156.
  • the calreticulin frameshift mutation produces a polypeptide comprising a sequence of SEQ ID NO: 157.
  • the at least one epitope derived from calreticulin comprising a frameshift mutation has a length of about 7 to 44 amino acids from SEQ ID NO: 157.
  • the at least one epitope comprises, consists essentially of, or consists of 7 to 11 amino acids from SEQ ID NO: 157.
  • the at least one epitope comprises, consists essentially of, or consists of 9 amino acids from SEQ ID NO: 157.
  • the at least one epitope comprises, consists essentially of, or consists of 15 amino acids from SEQ ID NO: 157.
  • the calreticulin frameshift mutation produces a polypeptide comprising a sequence of SEQ ID NO: 158.
  • the at least one epitope derived from calreticulin comprising a frameshift mutation has a length of about 7 to 44 amino acids from SEQ ID NO: 158.
  • the at least one epitope comprises, consists essentially of, or consists of 7 to 11 amino acids from SEQ ID NO: 158.
  • the at least one epitope comprises, consists essentially of, or consists of 9 amino acids from SEQ ID NO: 158.
  • the at least one epitope comprises, consists essentially of, or consists of 15 amino acids from SEQ ID NO:158.
  • the antigenic unit comprises a plurality of epitopes, each epitope independently selected from the group consisting of SEQ ID NOs: 4-11. In some embodiments, the antigenic unit comprises two or more epitopes that do not comprise the same sequence. In some embodiments, the antigenic unit comprises two or more epitopes that comprise the same sequence.
  • a protein or polypeptide as disclosed herein may comprise one or more linkers.
  • a protein or polypeptide of the disclosure, or a polynucleotide encoding the same comprises, from 5' to 3', a targeting unit, a dimerization unit, and an antigenic unit.
  • a linker connects one or more units.
  • a linker connects one or more subunits within a unit, such as the neoepitopes of the antigenic subunits described herein.
  • a linker may connect one or more cancer neoepitopes in an antigenic unit, e.g., one or more epitope(s) derived from calreticulin comprising a frameshift mutation.
  • a linker may connect an antigenic unit and a dimerization unit.
  • a linker may connect a dimerization unit and a targeting unit.
  • a linker may be a chemical bond, e.g., one or more covalent bonds or non- covalent bonds.
  • the linker is covalent.
  • the linker is non-covalent.
  • the linker comprises a peptide linker.
  • the peptide linker is between 2-30, 5-30, 10-30, 15-30, 20-30, 25-30, 2-25, 5- 25, 10-25, 15-25, 20-25, 2-20, 5-20, 10-20, 15-20, 2-15, 5-15, 10-15, 2-10, 5-10, or 2-5 amino acids in length, or greater than or equal to 2, 5, 10, 15, 20, 25, or 30 amino acids in length (and optionally up to 50, 40, 30, 25, 20, 15, 10, or 5 amino acids in length).
  • a linker may comprise flexible, rigid, and/or cleavable linkers described herein.
  • the linker includes at least one glycine, alanine, and serine amino acid to provide for flexibility.
  • the linker is a hydrophobic linker, such as including a negatively charged sulfonate group, polyethylene glycol (PEG) group, or pyrophosphate diester group.
  • a linker is cleavable to selectively release a moiety (e.g., a polypeptide) from a modulating agent, but is sufficiently stable to prevent premature cleavage.
  • Rigid linkers may be useful to keep a fixed distance between domains/moieties and to maintain their independent functions. Rigid linkers may also be useful when a spatial separation of domains is important for preserving the stability or bioactivity of one or more components in a fusion protein. Rigid linkers may have an alpha helix-structure or Pro-rich sequence, (XP) n , with X designating any amino acid, preferably Ala, Lys, or Glu.
  • XP alpha helix-structure or Pro-rich sequence
  • Cleavable linkers may release free functional domains in vivo.
  • linkers may be cleaved under specific conditions, such as in the presence of reducing reagents or proteases.
  • In vivo cleavable linkers may utilize the reversible nature of a disulfide bond.
  • a cleavable linker is a linker that includes a thrombinsensitive sequence (e.g., PRS) between the two Cys residues.
  • PRS thrombinsensitive sequence
  • In vitro thrombin treatment of a fusion protein comprising a CPRSC (SEQ ID NO: 172) sequence results in the cleavage of the thrombin-sensitive sequence, while a reversible disulfide linkage remains intact.
  • linkers are known and described, e.g., in Chen et al. 2013. Fusion Protein Linkers: Property, Design and Functionality. Adv Drug Deliv Rev. 65(10): 1357-1369.
  • In vivo cleavage of linkers in fusion proteins may also be carried out by proteases that are expressed in vivo under certain conditions, in specific cells or tissues, or constrained within certain cellular compartments. Specificity of many proteases offers slower cleavage of the linker in constrained compartments.
  • the cleavable linker may be a self-cleaving peptide linker, e.g., a T2A, P2A, E2A, or F2A peptide linker.
  • Example sequences of selfcleaving peptides include EGRGSLLTCGDVEENPGP (T2A) (SEQ ID NO: 173), ATNFSLLKQAGDVEENPGP (P2A) (SEQ ID NO: 174), QCTNYALLKLAGDVESNPGP (E2A) (SEQ ID NO: 175), and VKQTLNFDLLKLAGDVESNPGP (F2A) (SEQ ID NO: 176).
  • the linker may comprise a “ribosome skipping” sequence, e.g., a tPT2A linker.
  • Examples of molecules suitable for use in linkers described herein include a negatively charged sulfonate group; lipids, such as a poly (—CEE—) hydrocarbon chains, such as polyethylene glycol (PEG) group, unsaturated variants thereof, hydroxylated variants thereof, amidated or otherwise N-containing variants thereof; noncarbon linkers; carbohydrate linkers; phosphodiester linkers, or other molecule capable of covalently linking two or more components of a protein or polypeptide.
  • lipids such as a poly (—CEE—) hydrocarbon chains, such as polyethylene glycol (PEG) group, unsaturated variants thereof, hydroxylated variants thereof, amidated or otherwise N-containing variants thereof
  • PEG polyethylene glycol
  • Non-covalent linkers are also included, such as hydrophobic lipid globules to which the polypeptide is linked, for example through a hydrophobic region of a polypeptide or a hydrophobic extension of a polypeptide, such as a series of residues rich in leucine, isoleucine, valine, or perhaps also alanine, phenylalanine, or even tyrosine, methionine, glycine, or other hydrophobic residues.
  • Components of a protein or polypeptide may be linked using charge-based chemistry, such that a positively charged component of a protein or polypeptide is linked to a negative charge of another component.
  • the most hydrophobic antigenic subunit(s) may be substantially positioned in the middle of the antigenic unit and the most hydrophilic antigenic subunit(s) positioned at the beginning and/or end of the antigenic unit.
  • the neoepitopes may be arranged as alternating between a hydrophilic and a hydrophobic neoepitope.
  • Neoepitope hydrophilicity /hydrophobicity can be determined from the neoepitope sequence by persons of ordinary skill in the art. In general, Leu, He, Phe, Trp, Vai and Met are considered very hydrophobic; Cys, Tyr and Ala are considered hydrophobic; Thr, Glu, Gly, Ser, Gin, and Asp are considered neutral; and Arg, Lys, Asn, His, and Pro are considered hydrophilic.
  • the antigenic unit and the dimerization unit may be connected through a first linker.
  • the first linker may further comprise a restriction site to facilitate the construction of the polynucleotide.
  • the first linker is GLGGL (SEQ ID NO:98) or GLSGL (SEQ ID NO: 139).
  • a protein or polypeptide of the disclosure comprises one or more of a second linker between neoepitopes within the antigenic unit.
  • the number of second linkers is thus dependent on the number of neoepitopes present in the antigenic unit.
  • the second linker can be designed to be non-immunogenic, and may be a flexible linker, whereby the tumor neoepitopes, in spite of the high numbers of antigenic subunits present in the antigenic unit, are presented in an optimal manner to the T cells.
  • the second linker may be identical in all antigenic subunits. If, however, one or more of the neoepitopes comprises an amino acid motif similar to the linker, it may be an advantage to substitute the neighboring second linkers with a second linker of a different sequence. Also, if a neoepitope-second linker junction is predicted to constitute an epitope in itself, then a second linker of a different sequence might be used.
  • each second linker e.g., each linker between neoepitopes in the antigenic unit, comprises the same second linker sequence.
  • the antigenic unit comprises a plurality of second linkers linking neoepitope sequences, and individual second linkers in the plurality are not the same.
  • the length of a linker can be “short,” e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues, or “long,” e.g., at least 13 amino acid residues.
  • a linker is 10-50, 10-40, 10-30, 10-25, 10-20, 15-50, 15-40, 15-30, 15- 25, 15-20, 20-50, 20-40, 20-30, or 20-25 amino acid residues in length.
  • a linker comprises, consists essentially of, or consists of a (GS)n, (GGS)n, (GGGS)n (SEQ ID NO: 179), (GGSG)n (SEQ ID NO: 180), (GGSGG)n (SEQ ID NO: 181), and (GGGGS)n (SEQ ID NO: 182) sequence, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
  • a linker comprises, consists essentially of, or consists of an amino acid sequence selected from SEQ ID NOs:79-139, 165, or 169, as listed in Table 2.
  • a first linker as described herein may comprise, consist essentially of, or consist of an amino acid sequence selected from SEQ ID NOs:79-139, 165, or 169.
  • a second linker as described herein may comprise, consist essentially of, or consist of an amino acid sequence selected from SEQ ID NOs:79-139, 165, or 169.
  • GSAT SEQ ID NO: 140
  • SEG linkers include, for example and without limitation, GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 167).
  • Other suitable linkers such as the T cell epitope linker ELKTPLGDTTHT (SEQ ID NO: 168), are known in the art, and described, for example, in WO 2022/233851.
  • the polypeptide or protein of the disclosure leads to attraction of dendritic cells (DCs), neutrophils, and other immune cells.
  • DCs dendritic cells
  • the polypeptide or protein comprising the targeting unit can not only target the antigens to specific cells, but can also facilitate a response-amplifying effect (adjuvant effect) by recruiting specific immune cells to the administration site of the pharmaceutical composition.
  • adjuvant effect a response-amplifying effect
  • the term “targeting unit” as used herein can refer to a unit that delivers the polypeptide or protein with an antigen of the disclosure, e.g., a neoepitope derived from calreticulin, to an antigen presenting cell for MHC class Il-restricted presentation to CD4+ T cells, or for providing cross presentation to CD8+ T cells by MHC class I restriction.
  • the targeting unit may be connected through the dimerization unit to the antigenic unit, wherein the latter is in either the COOH-terminal or the NH2 -terminal end of the polypeptide or protein.
  • the antigenic unit is in the COOH-terminal end of the polypeptide or protein.
  • the targeting unit is designed to target the polypeptide or protein of the disclosure to surface molecules expressed on the relevant antigen presenting cells (APC), such as molecules expressed exclusively on subsets of dendritic cells (DC).
  • APC relevant antigen presenting cells
  • the targeting unit has affinity for an MHC class II protein.
  • the targeting unit comprises, consists essentially of, or consists of an antibody-binding region, such as the antibody variable domains (VL and VH) with specificity for MHC class II proteins, for example selected from the group consisting of anti- HLA-DP, anti-HLA-DR and anti-pan HLA class II.
  • VL and VH antibody variable domains
  • the dimerization unit comprises, consists essentially of, or consists of hinge exons hl and h4 connected through a linker to a CH3 domain of human IgG3.
  • the linker that connect the hinge region and another domain that facilitate dimerization, such as an immunoglobulin domain is a G3S2G3SG (SEQ ID NO: 169) linker.
  • the dimerization unit may have any orientation with respect to the antigenic unit and the targeting unit.
  • the antigenic unit is in the COOH-terminal end of the dimerization unit with the targeting unit in the N-terminal end of the dimerization unit.
  • the dimerization unit comprises, consists of, or consists essentially of ELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNI FSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 178).
  • the polynucleotide further comprises a nucleotide sequence encoding a signal peptide.
  • the signal peptide is included to facilitate secretion of the polypeptide encoded by the polynucleotide of the disclosure in the cells transfected with said polynucleotide.
  • a signal peptide comprises, consists of, or consists essentially of a signal peptide of human MIP-lu.
  • the signal peptide from human MIP-lu comprises, consists of, or consists essentially of MQVSTAALAVLLCTMALCNQVLS (SEQ ID NO: 163).
  • nucleotide sequence encoding such human MIP-lu comprises, consists of, or consists essentially of 5' - ATGCAGGTCTCCACTGCTGCCCTTGCCGTCCTCCTCTGCACCATGGCTCTCTGCAACCAGGTCCTCTCT- 3 ' (SEQ ID NO: 186).
  • the nucleotide sequence comprises a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 186.
  • nucleotide sequence comprises a sequence of SEQ ID NO: 186 or a sequence with 1, 2, 3, 4 or 5 insertions, substitutions or deletions relative thereto.
  • a dimeric protein or polypeptide of the disclosure comprises MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQI PQNFIADYFETSSQCSKPSVI FLTKRGRQVCA DPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNI FSCSVMHEALHNR FTQKSLSLSPGK (SEQ ID NO: 170).
  • the disclosure further relates to polypeptides comprising the antigenic units and targeting units described above.
  • the polypeptides comprise an antigenic unit, a dimerization unit, and a targeting unit.
  • the polypeptides comprise an antigenic unit, a dimerization unit, a targeting unit and a signal peptide.
  • the polypeptides form a dimer.
  • polypeptides may be expressed in vitro for production of a pharmaceutical composition comprising the polypeptides or proteins according to the disclosure, or the polypeptide may be expressed in vivo as a result of administration of a pharmaceutical composition comprising a polynucleotide encoding the polypeptide as defined above.
  • dimeric proteins are formed when polypeptides comprising the dimerization unit are expressed.
  • the dimeric protein may be a homodimer, z.e., wherein the two polypeptide chains are identical and consequently comprise identical neoepitopes, or the dimeric protein may be a heterodimer comprising two different monomeric polypeptides encoded in the antigenic units. The latter may be relevant if the amount of neoepitopes exceeds an upper size limit for the antigenic unit.
  • the protein is a dimeric protein. In some embodiments, the dimeric protein is a homodimeric protein. In some embodiments, the dimeric protein is a heterodimeric protein.
  • the polypeptide comprises, consists essentially of, or consists of
  • the dimeric protein comprises, consists essentially of, or consists of two polypeptides, each comprising MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQI PQNFIADYFETSSQCSKPSVI FLTKRGRQVCA DPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNI FSCSVMHEALHNR FTQKSLSLSPGKGLGGLRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTEA (SEQ ID NO: 177).
  • the dimeric protein comprises, consists essentially of, or consists of two polypeptides, each consisting of MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQI PQNFIADYFETSSQCSKPSVI FLTKRGRQVCA DPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNI FSCSVMHEALHNR FTQKSLSLSPGKGLGGLRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTEA (SEQ ID NO: 177).
  • Polynucleotides encoding the proteins or polypeptides described herein, or fragments thereof, may be incorporated into a vector.
  • Vectors including those derived from retroviruses such as lentivirus, are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene, and its propagation in daughter cells.
  • suitable vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • an expression vector may be provided to a cell in the form of a viral vector.
  • Viruses that are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers.
  • the disclosure provides a vector comprising a nucleotide sequence as defined above.
  • the vector allows for easy exchange of the various units described above, in particularly the antigenic unit.
  • the expression vector may be pUMVC4a vector (comprising a CMV promoter, a CMV intron, and a beta globin polyA signal) or vectors based on a NTC9385R (NanoplasmidTM) vector backbone. Suitable vectors are described in WO2017/118695, the contents of which are incorporated by reference herein.
  • the antigenic unit may be exchanged with an antigenic unit cassette restricted by the Sfil restriction enzyme cassette where the 5' site is incorporated in the GLGGL (SEQ ID NO:98) or GLSGL (SEQ ID NO: 139) linker, e.g., first linker, and the 3' site is included after the stop codon in the vector.
  • An example, non-limiting nucleotide sequence encoding such first linker, e.g., GLGGL (SEQ ID NO:98) comprises, consists of, or consists essentially of 5' - GGCCTCGGTGGCCTG - 3' (SEQ ID NO: 187).
  • the vector comprises a promoter sequence operably linked to a sequence encoding the protein, polypeptide or fragment thereof described supra.
  • the promoter is constitutive.
  • the promoter is tissue or cell type specific.
  • the promoter comprises a Cytomegalovirus (CMV) promoter.
  • the vector comprises one or more sequences to enhance expression of the protein, polypeptide or fragment thereof, including, but not limited to, a sequence encoding an intron, polyadenylation signal, or Woodchuck hepatitis virus (WHV) posttranscriptional regulatory element (WPRE). Selection of suitable vector elements will be known to one of ordinary skill in the art.
  • the disclosure provides polynucleotides encoding the polypeptides described above.
  • the polynucleotide may comprise a DNA nucleotide sequence or an RNA nucleotide sequence, such as genomic DNA, cDNA, and RNA sequences, either double stranded or single stranded.
  • the polynucleotide is optimized to the species to express the polypeptide according to the disclosure, z.e., human codon optimized.
  • polynucleotides encoding a protein or polypeptide of the present disclosure.
  • the polynucleotide comprises a sequence encoding an antigenic unit and a sequence encoding a targeting unit.
  • the polynucleotide comprises a sequence encoding an antigenic unit, a dimerization unit, and a targeting unit.
  • the polynucleotide comprises a sequence encoding an antigenic subunit comprising one or more neoepitopes derived from calreticulin comprising a frameshift mutation, optionally wherein the neoepitopes are linked via linkers, a dimerization unit comprising an immunoglobulin constant domain, and a targeting domain comprising a MIP-lu sequence.
  • the polynucleotide comprises a sequence encoding an antigenic subunit comprising one or more neoepitopes each comprising 7 to 44 amino acids of any one of SEQ ID NOs:2, 3, or 141-158; a sequence encoding a dimerization unit comprising a hinge exon hl from IgG3, a hinge exon h4 from IgG3, a dimerization unit linker, and human CH3 domain of IgG3, e.g., SEQ ID NO: 178; and a sequence encoding a targeting domain comprising a MIP-lu sequence, e.g., SEQ ID NO:159.
  • the polynucleotide comprises a sequence encoding an antigenic subunit comprising one or more neoepitopes each comprising 7 to 44 amino acids of any one of SEQ ID NOs:2, 3, or 141-158; a sequence encoding a dimerization unit comprising a hinge exon hl from IgG3, a hinge exon h4 from IgG3, a dimerization unit linker, and human CH3 domain of IgG3 of SEQ ID NO: 170; and a sequence encoding a targeting domain comprising a MIP-lu sequence of SEQ ID NO: 159.
  • the polynucleotide comprises a sequence encoding an antigenic unit, a sequence encoding a dimerization unit and a sequence encoding a targeting unit, wherein the sequences of the antigenic unit and the dimerization unit and/or the dimerization unit and the targeting unit are separated via sequences encoding linkers as described herein.
  • a protein or polypeptide of the present disclosure may be provided to a subject via a pharmaceutical composition comprising a polynucleotide encoding the same, wherein the polynucleotide is associated with sufficient other sequences to achieve expression in a system of interest (e.g., in a particular cell type, tissue, organism, etc.).
  • the polynucleotides that encode the protein or polypeptide, or fragment thereof may be operationally associated with one or more replication, integration, and/or expression signals appropriate and/or sufficient to achieve integration, replication, and/or expression of the provided nucleic acid in a system of interest (e.g., in a particular cell, tissue, organism, etc.).
  • the polynucleotides may be or may include DNA, RNA, or any other nucleic acid moiety or entity as described herein, and may be prepared by any technology described herein or otherwise available in the art (e.g., synthesis, cloning, amplification, in vitro synthesis, replication in host cell such as E. coh. or in vivo transcription, etc.).
  • the polynucleotide sequence may include, for example and without limitation, DNA, RNA, modified oligonucleotides (e.g., chemical modifications, such as modifications that alter the backbone linkages, sugar molecules, and/or nucleic acid bases), and artificial nucleic acids.
  • modified oligonucleotides e.g., chemical modifications, such as modifications that alter the backbone linkages, sugar molecules, and/or nucleic acid bases
  • artificial nucleic acids e.g., DNA, RNA, modified oligonucleotides (e.g., chemical modifications, such as modifications that alter the backbone linkages, sugar molecules, and/or nucleic acid bases), and artificial nucleic acids.
  • provided polynucleotides encoding a protein or polypeptide, or polypeptide fragments thereof may be operationally associated with one or more replication, integration, and/or expression signals appropriate and/or sufficient to achieve integration, replication, and/or expression of the provided nucleic acid in a system of interest (e.g., in a particular cell, tissue, organism, etc.).
  • a cell may be used to express a protein or polypeptide, e.g., VaccibodyTM, described herein.
  • a cell e.g., cell line
  • a cell comprises a polypeptide encoding a protein or polypeptide, e.g., VaccibodyTM, described herein.
  • the disclosure also relates to a host cell comprising a polynucleotide sequence as defined above, or comprising a vector as defined above for expression of the polypeptide according to the disclosure.
  • the present disclosure is further directed, in part, to pharmaceutical compositions comprising one or more polynucleotides of the disclosure, e.g., a VaccibodyTM comprising at least one epitope derived from calreticulin, as described herein.
  • the present disclosure is further directed, in part, to pharmaceutical compositions comprising one or more proteins or polypeptides of the disclosure as described herein.
  • the pharmaceutical composition comprises one or more pharmaceutically acceptable adjuvants.
  • compositions include, but are not limited to poly-ICLC, 1018 ISS, aluminum salts, Amplivax, AS 15, BCG, CP- 870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFactEVl P321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK- 432, OM-174, OM-197-MP-EC, ONTAK, PLGA microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta- glucan, Pam3Cys, Aquila's QS21 stimulon, vadimezan, and/or AsA404
  • the pharmaceutical composition comprises a pharmaceutically acceptable amphiphilic block co-polymer.
  • the amphiphilic block co-polymer comprises blocks of poly(ethylene oxide) and polypropylene oxide).
  • An “amphiphilic block co-polymer” as used herein is a linear or branched copolymer comprising or consisting of blocks of poly(ethylene oxide) (“PEO”) and blocks of polypropylene oxide) (“PPO”).
  • PEO poly(ethylene oxide)
  • PPO polypropylene oxide
  • Typical examples of useful PEO-PPO amphiphilic block copolymers have the general structures PEO-PPO-PEO (poloxamers), PPO PEO PPO, (PEO PPO-) 4ED (a poloxamine), and (PPO PEO-)4ED (a reverse poloxamine), where “ED” is an ethylenediaminyl group.
  • a “poloxamer” is a linear amphiphilic block co-polymer constituted by one block of poly(ethylene oxide) coupled to one block of polypropylene oxide) coupled to one block of PEO, z.e., a structure of the formula EOa-POb-EOa, where EO is ethylene oxide, PO is propylene oxide, a is an integer from 2 to 130, and b is an integer from 15 to 67.
  • Poloxamers are conventionally named by using a 3 -digit identifier, where the first 2 digits multiplied by 100 provides the approximate molecular mass of the PPO content, and where the last digit multiplied by 10 indicates the approximate percentage of PEO content.
  • Polyxamer 188 refers to a polymer comprising a PPO block of a molecular weight of about 1800 (corresponding to b being about 31 PPO) and approximately 80% (w/w) of PEO (corresponding to a being about 82).
  • the values are known to vary to some degree, and commercial products such as the research grade Lutrol® F68 and the clinical grade Kolliphor®.
  • Pol oxamer 188 exhibits a large variation in molecular weight (between
  • a “poloxamine” or “sequential poloxamine” (commercially available under the trade name of Tetronic®) is an X-shaped block co-polymers that bears four PEO-PPO arms connected to a central ethylenediamine moiety via bonds between the free OH groups comprised in the PEO-PPO-arms and the primary amine groups in ethylenediamine moiety.
  • Reverse poloxamines are likewise X- shaped block co-polymers that bear four PPO-PEO arms connected to a central ethylenediamine moiety via bonds between the free OH groups comprised in the PPO-PEO arms and the primary amine groups in ethylenediamine.
  • Tetronic® 704 has a total average molecular weight of 5500, a total average weight of PPO units of 3300, and a PEO percentage of about 40%; and Tetronic® 304 has a total average molecular weight of 1650, a total average weight of PPO units of 990, and a PEO percentage of about 40%.
  • the composition comprises the amphiphilic block copolymer in an amount of from 0.2% weight per volume (w/v) to 20% w/v, such as from 0.2% w/v to 18% w/v, 0.2% w/v to 16% w/v, 0.2% w/v to 14% w/v, 0.2% w/v to 12% w/v, 0.2% w/v to 10% w/v, 0.2% w/v to 8% w/v, 0.2% w/v to 6% w/v, 0.2% w/v to 4% w/v, 0.4% w/v to 18% w/v, 0.6% w/v to 18% w/v, 0.8% w/v to 18% w/v, 1% w/v to 18% w/v, 2% w/v to 18% w/v, 1% w/v to 5% w/v, or 2% w/v to 4% w/w/
  • the pharmaceutical composition comprises a polynucleotide, e.g., comprised in a vector such as apolycistronic vector, and is administered by intramuscular or intradermal injection.
  • a polynucleotide e.g., comprised in a vector such as apolycistronic vector, and is administered by intramuscular or intradermal injection.
  • the pharmaceutical composition of the disclosure may comprise the polynucleotide in a range of from 0.1 to 10 mg per unit dosage, e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 mg or e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg.
  • the pharmaceutical composition of the disclosure typically comprises the polypeptide or protein in the range of from 5 pg to 5 mg.
  • the amount of polynucleotide, polypeptide, or protein may vary depending on whether the pharmaceutical composition is administered for prophylactic, to reduce the likelihood of disease, or for therapeutic treatment, the severity of the disease in individuals which are infected, and on parameters like the age, weight, gender, medical history and preexisting conditions.
  • compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g, those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; trans-dermally; or nasally, pulmonary, and/or to other mucosal surfaces.
  • oral administration for example, drenches (aqueous or non-aqueous solutions
  • compositions are involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic s
  • salts include, but are not limited to, nontoxic acid addition salts, which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods used in the art such as ion exchange.
  • nontoxic acid addition salts which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods used in the art such as ion exchange.
  • pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate,
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate, and aryl sulfonate.
  • compositions described herein with a pharmaceutically acceptable excipient.
  • Pharmaceutically acceptable excipient includes an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
  • compositions may be made following conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing, and filling for hard gelatin capsule forms.
  • a preparation can be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous solution or suspension.
  • Such a liquid formulation may be administered directly per os.
  • compositions may be formulated for delivery to a cell and/or to a subject via any route of administration.
  • Modes of administration to a subject may include injection, infusion, inhalation, intranasal, intraocular, topical delivery, inter-cannular delivery, or ingestion.
  • Injection includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intra-cerebrospinal, and intra-stemal injection and infusion.
  • administration is intramuscular.
  • Intramuscular administration can be performed with a needle, or it can be needle-free.
  • intramuscular injection is performed by a needle-free hypodermic injector system, e.g., a PharmaJetTM.
  • administration includes aerosol inhalation, e.g., with nebulization.
  • administration is systemic (e.g., oral, rectal, nasal, sublingual, buccal, or parenteral), enteral (e.g., system -wide effect, but delivered through the gastrointestinal tract), or local (e.g., local application on the skin, or intravitreal injection).
  • one or more compositions is administered systemically.
  • administration may be a single dose.
  • administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic e.g., individual doses separated by a common period of time) dosing.
  • administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.
  • six, eight, ten, 12, 15 or 20 or more administrations may be given to the subject during one treatment or over a period of time as a treatment regimen.
  • administrations may be given as needed, e.g., for as long as symptoms associated with the disease, disorder or condition persist. In some embodiments, repeated administrations may be indicated for the remainder of the subject’s life. Treatment periods may vary and could be, e.g., one day, two days, three days, one week, two weeks, one month, two months, three months, six months, a year, or longer.
  • administration comprises a series of doses, e.g., a loading dose followed by a maintenance dose.
  • a loading dose can be administered to achieve therapeutically effective levels of a pharmaceutical composition of the disclosure, with administration being at higher dose or higher frequency than a maintenance dose.
  • the loading dose is administered more frequently than the maintenance dose.
  • the loading dose is administered at higher dose level than the maintenance dose.
  • the loading dose is administered at an equal dose level to the maintenance dose. .
  • a loading dose is administered once, twice, three times, four times, five times, or more than five times.
  • a maintenance dose is administered once, twice, three times, four times, five times, or more than five times.
  • the loading dose and maintenance dose are about 3 mg, about 6 mg, or about 9 mg. In some embodiments, the loading dose and maintenance dose are both about 3 mg.
  • the loading dose is administered weekly (QW). In some embodiments, the loading dose is administered biweekly (Q2W). In some embodiments, the loading dose is administered every 3 weeks (Q3W). In some embodiments, the loading dose is administered monthly or every 4 weeks (Q4W). In some embodiments, the maintenance dose is administered biweekly (Q2W). In some embodiments, the maintenance dose is administered every 3 weeks (Q3W). In some embodiments, the maintenance dose is administered monthly or every 4 weeks (Q4W). In some embodiments, the maintenance dose is administered every 6 weeks (Q6W). In some embodiments, the maintenance dose is administered every 12 weeks (Q12W).
  • the loading dose is administered bi-weekly, e.g., for five doses, and the maintenance dose is administered every 6 weeks.
  • administration is at about 3 mg, about 6 mg, or about 9 mg, by intramuscular injection, e.g., needle-free injection.
  • the loading dose is administered every 3 weeks, e.g., for four doses, and the maintenance dose is administered every 6 weeks.
  • administration is at about 3 mg, about 6 mg, or about 9 mg, by intramuscular injection, e.g., needle-free injection.
  • the loading dose is administered every 3 weeks , e.g., for four doses, and the maintenance dose is administered every 12 weeks.
  • administration is at about 3 mg, about 6 mg, or about 9 mg, by intramuscular injection, e.g., needle-free injection.
  • the disclosure relates in part to methods for preparing a pharmaceutical composition comprising an immunologically effective amount of a polynucleotide of the disclosure, or a protein or polypeptide encoded thereby, in vitro.
  • in vitro synthesis may be carried out by any suitable method known to the person skilled in the art, such as through peptide synthesis or expression of the polypeptide in any of a variety of expressions systems followed by purification.
  • Methods of producing pharmaceutical compositions comprising the polypeptides or proteins of the disclosure include, for example and without limitation, a) transfecting a cell population with a polynucleotide disclosed herein; b) culturing the cell population; c) collecting and purifying the protein or the polypeptide expressed by the cell population, and d) mixing the protein or polypeptide obtained under step c) with a pharmaceutically acceptable carrier, thereby obtaining the pharmaceutical composition.
  • a buffer may be added to the pharmaceutical composition.
  • a buffer may be added to the pharmaceutical composition.
  • an adjuvant may be added to the pharmaceutical composition.
  • Purification may be carried out according to any suitable method known in the art, such as chromatography, centrifugation, or differential solubility.
  • nucleotide sequences encoding the targeting unit, the dimerization unit, the subunits of the antigenic unit may be synthesized individually and then ligated together, to produce the final polynucleotide.
  • the final polynucleotide is introduced into a vector backbone.
  • the methods described supra are preceded by a method of identifying the neoepitopes to be included in the polynucleotide.
  • This method of identifying the neoepitopes can include the steps of a) sequencing the genome or exome of a tumor, or otherwise determining the status of a calreticulin mutation in a patient’s tumor, b) identifying the calreticulin neoepitopes from the tumor, and c) selecting the neoepitope or a combination thereof based on predicted antigenicity.
  • sequencing the tumor may be by through any suitable method, such as by obtaining a biopsy of the tumor, or by excision of the tumor, or from collection of any suitable body fluid, such as a blood sample or a urine sample.
  • the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, adjuvant, or buffer.
  • a pharmaceutical composition of the disclosure may be formulated into any suitable formulation, such as a liquid formulation for intradermal or intramuscular injection.
  • a pharmaceutical composition of the disclosure may be administered in any suitable route of administration for either a polypeptide/protein vaccine or a polynucleotide vaccine, such as administration by injection intradermally, intramuscular, subcutaneously, or by mucosal or epithelial application, such as intranasally, orally, enteral or to the bladder.
  • administration may be intramuscular or intradermal. In some embodiments, administration is intramuscular. Intramuscular administration can be performed with a needle, or it can be needle-free. In some embodiments, intramuscular injection is performed by a needle-free hypodermic injector system, e.g., a PharmaJetTM.
  • a needle-free hypodermic injector system e.g., a PharmaJetTM.
  • the pharmaceutical composition is administered by intranodal injection.
  • intranodal injection means that the pharmaceutical composition is injected into the lymph nodes.
  • administration is bronchial, buccal, dermal, enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal, vaginal, vitreal, etc.
  • a specific organ e.g., intrahepatic
  • mucosal nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal, vaginal, vitreal, etc.
  • the pharmaceutical composition is administered as a single dose.
  • the pharmaceutical composition is administered using intermittent and/or periodic dosing.
  • administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.
  • six, eight, ten, 12, 15, or 20 or more administrations may be given to the subject during one treatment or over a period of time as a treatment regimen.
  • Pharmaceutical compositions according to the present disclosure may be delivered in a therapeutically effective amount.
  • a precise therapeutically effective amount is an amount of a composition that will yield the most effective results in terms of efficacy of treatment in a given subject.
  • This amount will vary depending upon a variety of factors, including but not limited to characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), physiological condition of a subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), nature of a pharmaceutically acceptable carrier or carriers in a formulation, and/or route of administration.
  • characteristics of the therapeutic compound including activity, pharmacokinetics, pharmacodynamics, and bioavailability
  • physiological condition of a subject including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication
  • nature of a pharmaceutically acceptable carrier or carriers in a formulation and/or route of administration.
  • the polynucleotides, polypeptides, and proteins of the disclosure are for methods of use in the treatment of cancer.
  • the cancer comprises a myeloproliferative neoplasm.
  • the polynucleotides, polypeptides and dimeric proteins may be formulated in a pharmaceutical composition as described herein.
  • Such pharmaceutical composition may be used, e.g., as a vaccine, e.g., a cancer vaccine.
  • the methods described herein it is possible to treat a patient suffering from cancer by examining any mutations present in the tumor in the patient, producing the pharmaceutical composition, e.g., vaccine, e.g., vaccine comprising a calreticulin VaccibodyTM as disclosed herein, and then immunizing the patient with the vaccine directed exactly to neoantigens present in his or her tumor, e.g., directed against calreticulin frameshift neoantigens.
  • the cancer may be a primary tumor, metastasis or both.
  • the tumor examined for mutations may be a primary tumor or a metastasis.
  • the treatment comprises administering a pharmaceutical composition comprising a polynucleotide as described herein.
  • the patient is treated with a pharmaceutical composition, e.g., vaccine, comprising a combination of peptides, e.g., directed against calreticulin frameshift neoantigens.
  • a pharmaceutical composition e.g., vaccine
  • a combination of peptides e.g., directed against calreticulin frameshift neoantigens.
  • the pharmaceutical composition when the pharmaceutical composition comprises a polynucleotide of the disclosure, the pharmaceutical composition is administered intramuscularly.
  • a pharmaceutical composition of the disclosure be administered as a single dosage, or may be administered in repeated doses. When administration is repeated, subsequent doses may be administered with at least 3-week intervals between dosing, to avoid exhaustion of the T cells.
  • the dosage regimen would be vaccination week 0, 3, 6 and then every 4 weeks as long as the patient has clinical benefit.
  • the pharmaceutical composition may be administered for at least a year.
  • the pharmaceutical composition is administered in an immunologically effective amount.
  • immunologically effective amount is meant the amount required to establish a tumor reducing effect.
  • the physician determines the dosage that typically is in the range of 0.3-6 mg for DNA-based compositions, and in the range of 5 pg-5 mg for polypeptide/protein compositions.
  • the cancer is a myeloproliferative neoplasm.
  • the myeloproliferative neoplasm is chronic myeloid leukemia, acute myeloid leukemia, polycythaemia vera, essential thrombocythemia, primary myelofibrosis, chronic eosinophilic leukemia, chronic myelomonocytic leukemia, systemic mastocytosis, idiopathic myelofibrosis, or myeloma.
  • the cancer is a hematologic malignancy.
  • the hematologic malignancy is a leukemia.
  • the leukemia may be acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), myelodysplasia, myelodysplastic syndromes, acute T-lymphoblastic leukemia, or acute promyelocytic leukemia, chronic myelomonocytic leukemia, or myeloid blast crisis of chronic myeloid leukemia.
  • the cancer is a myeloproliferative neoplasm (MPN).
  • MPN myeloproliferative neoplasm
  • the MPN is chronic myeloid leukemia, acute myeloid leukemia, polycythaemia vera, essential thrombocythemia, primary myelofibrosis, chronic eosinophilic leukemia, chronic myelomonocytic leukemia, systemic mastocytosis, idiopathic myelofibrosis, or myeloma.
  • the cancer may be a vascularized tumor, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, neuroblastoma, sarcoma e.g., an angiosarcoma or chondrosarcoma), larynx cancer, parotid cancer, biliary tract cancer, thyroid cancer, acral lentiginous melanoma, actinic keratoses, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, anal canal cancer, anal cancer, anorectum cancer, astrocytic tumor, Bartholin gland carcinoma, basal cell carcinoma, biliary cancer, bone cancer, bone marrow cancer, bronchial cancer, bronchial gland carcinoma, carcinoid, cholangiocarcinoma, cho
  • compositions of the present disclosure may be combined with any other suitable treatment for cancer.
  • the patient when the patient is administered a composition of the present disclosure for treatment of a cancer, the patient is further administered a radiation therapy, chemotherapy, surgical treatment, or combination thereof.
  • Example therapeutic agents that may be used as part of a combination therapy in treating cancer, include, for example and without limitation, radiation, mitomycin, tretinoin, ribomustin, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin, nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptinium acetate, ketanserin, doxifluridine, etretinate, isotretinoi
  • the enriched HLA-bound peptides were subsequently analyzed using an advanced Orbitrap Fusion Lumos® mass spectrometer, which was coupled to a nanoLC system.
  • the raw mass spectrometry (MS) data files were then searched with the UniProtKB (Uniprot knowledgebase release 2020_0420).
  • Example 2 CALR MUT -specific T cells were detected in the peripheral blood of myeloproliferative neoplasm (MPN) patients harboring the CALR exon 9+1 frameshift mutation
  • PBMCs from MPN patients carrying CALR MUT or a healthy donor were cultured in the presence of a peptide pool derived from CALR MUT or DMSO.
  • Peptide pools are provided in Tables 3 and 4 for calreticulin frameshift mutation and wildtype peptides, respectively.
  • amino acids in italic are found in wild type CALR.
  • Amino acids indicated in bold are the core sequence (36 amino acids in total) found in all MPN patients carrying CALR exon9 frameshift mutants.
  • Expanded PBMC cultures were re-stimulated by autologous B cells pulsed with the CALR MUT peptide pool used for T cell expansion.
  • expanded PBMC cultures were re-stimulated with B cells treated with DMSO or a peptide pool derived from wild-type CALR (CALR WT ).
  • expanded PBMC cultures were re-stimulated with B cells pulsed with a peptide pool derived from common viral proteins (CEF: CMV, EBV, and Flu).
  • CEF common viral proteins
  • Example 3 MPN antigen-specific T cells were expanded from healthy donor naive T cells and were detectable in the human T Cell repertoire.
  • Naive T cells from healthy donors were expanded with a peptide pool derived from CALR MUT as described supra.
  • Naive T cells co-cultured with moDC-treated with DMSO were cultured in parallel as a negative control.
  • Pre-expanded T cells were restimulated by CD2-depleted autologous PBMCs pulsed with the indicated peptide pools or DMSO.
  • IFNy-producing T cells were detected by IFNy ELISpot. Representative ELISpot analysis is shown in FIG. 3B, and summarized data is shown in FIG. 3C, for CALR MUT - specific T cells expanded from healthy donor naive T cells.
  • CALR MUT -specific T cell expansion can be induced from naive T cells.
  • Example 4 VaccibodiesTM encoding MPN neoantigens elicited CALR-specific T cell responses in C57BL/6 mice.
  • the HPV16 E6 E7 pDNA is a positive control encoding the full protein.
  • 20 pg or 60 pg of CALR-44mer VaccibodyTM or appropriate controls were administered intramuscularly followed by electroporation around the injection site at day 7 and boosted at day 14 post tumor implantation.
  • the spleens were harvested and processed into single cell suspensions, restimulated with a 15-mer peptide pool or DMSO for 24 h, and assessed for antigen specific T cell responses by IFNY enzyme linked immune absorbent spots (ELISpots), shown in FIG. 4.
  • IFNY enzyme linked immune absorbent spots IFNY enzyme linked immune absorbent spots
  • Example 5 An engineered mouse tumor cell line, TC-1, and was functionally regressed with therapeutic MPN VaccibodyTM immunization.
  • TC-1 a C57BL/6 mouse lung epithelial cell line
  • This neoantigen is present in CALR MUT patients carrying insertion/deletion (indels) mutations in exon 9, which result in a frameshift and the generation of a new C-terminal of the calreticulin protein containing the conserved 44mer neoantigen.
  • the expression construct used to engineer this cell line included an HA-tagged peptide ubiquitin cassette for CALR frameshift mutation and Blasticidin resistance gene.
  • the cells were transduced with a pLVX lentivirus using Lenti-X packaging single shot (VSV-G; ClontechTM, #631276).
  • the transduced cells were positively selected via Blasticidin selection, single cell sorted, and expanded.
  • the expanded single clone populations were validated by intracellular staining with Alexa Fluor 647 conjugated HA-tag antibody (R&D SystemsTM, Cat# IC6875R) using flow cytometry, as shown in FIG. 5A.
  • a total of about 2.5xl0 5 engineered TC-1 cells were implanted subcutaneously on the right flank of each mouse. Five mice were used for each group. Seven days post-tumor implantation, 2, 20 or 60 pg of pDNA vaccines (CALR-44mer VaccibodyTM carrying the CALR44mer neoantigen (SEQ ID NO: 3) or empty VaccibodyTM without the 44mer neoantigen) were injected intramuscularly and electroporated into the mouse right upper leg. The immunization was followed by a booster dose on day 14. Tumor growth was monitored twice weekly over a 40-day period.
  • mice that received two vaccinations with CALR44mer VaccibodyTM pDNA demonstrated significant tumor regression in a dosedependent manner. The most pronounced tumor regression was observed in animals vaccinated with a 20 pg dose, which resulted in an approximately 65% reduction in tumor size in average post-vaccine boost.
  • Immunization with CALR-44mer VaccibodyTM elicits potent anti -turn or protection in a dose response manner in mice transplanted with TC-1 engineered cells.

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)
  • Zoology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Animal Behavior & Ethology (AREA)
  • Molecular Biology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Toxicology (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Oncology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des acides nucléiques codant pour des protéines de fusion recombinantes ciblant la calréticuline, ainsi que des compositions pharmaceutiques comprenant de tels acides nucléiques et des méthodes thérapeutiques utilisant de tels acides nucléiques et compositions pharmaceutiques, tel que pour le traitement du cancer.
PCT/US2025/021343 2024-03-26 2025-03-25 Compositions et méthodes pour le traitement de néoplasmes myéloprolifératifs Pending WO2025207632A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463569841P 2024-03-26 2024-03-26
US63/569,841 2024-03-26

Publications (1)

Publication Number Publication Date
WO2025207632A1 true WO2025207632A1 (fr) 2025-10-02

Family

ID=95477379

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/021343 Pending WO2025207632A1 (fr) 2024-03-26 2025-03-25 Compositions et méthodes pour le traitement de néoplasmes myéloprolifératifs

Country Status (1)

Country Link
WO (1) WO2025207632A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017118695A1 (fr) 2016-01-08 2017-07-13 Vaccibody As Vaccin thérapeutique anticancéreux à néoépitope
EP3494985B1 (fr) * 2013-09-16 2021-02-24 CeMM - Forschungszentrum für Molekulare Medizin GmbH Composition de vaccin comprenant de la calréticuline mutante
WO2022233851A1 (fr) 2021-05-03 2022-11-10 Nykode Therapeutics ASA Constructions immunogènes et vaccins destinés à être utilisés dans le traitement prophylactique et thérapeutique de maladies infectieuses
WO2022238363A1 (fr) 2021-05-10 2022-11-17 Nykode Therapeutics ASA Constructions immunogènes et vaccins destinés à être utilisés dans le traitement prophylactique et thérapeutique de maladies infectieuses

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3494985B1 (fr) * 2013-09-16 2021-02-24 CeMM - Forschungszentrum für Molekulare Medizin GmbH Composition de vaccin comprenant de la calréticuline mutante
WO2017118695A1 (fr) 2016-01-08 2017-07-13 Vaccibody As Vaccin thérapeutique anticancéreux à néoépitope
WO2022233851A1 (fr) 2021-05-03 2022-11-10 Nykode Therapeutics ASA Constructions immunogènes et vaccins destinés à être utilisés dans le traitement prophylactique et thérapeutique de maladies infectieuses
WO2022238363A1 (fr) 2021-05-10 2022-11-17 Nykode Therapeutics ASA Constructions immunogènes et vaccins destinés à être utilisés dans le traitement prophylactique et thérapeutique de maladies infectieuses

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
"NCBI", Database accession no. NG_ 029662.1
"Uniprot", Database accession no. P27797
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", 1992, GREENE PUBLISHING ASSOCIATES
CHEN ET AL.: "Fusion Protein Linkers: Property, Design and Functionality", ADV DRUG DELIV REV., vol. 65, no. 10, 2013, pages 1357 - 1369, XP028737352, DOI: 10.1016/j.addr.2012.09.039
GIGOUX MATHIEU ET AL: "Calreticulin mutant myeloproliferative neoplasms induce MHC-I skewing, which can be overcome by an optimized peptide cancer vaccine", SCIENCE TRANSLATIONAL MEDICINE(SUPPLEMENTARY MATERIALS), vol. 14, no. 649, 15 June 2022 (2022-06-15), XP093134601, ISSN: 1946-6234, DOI: 10.1126/scitranslmed.aba4380 *
HIGGINS D.THOMPSON J.GIBSON T.THOMPSON J.D.HIGGINS D.G.GIBSON T.J.: "CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice", NUCLEIC ACIDS RES., vol. 22, 1994, pages 4673 - 4680, XP002956304
MARTIN: "Remington's Pharmaceutical Sciences", 1975, MACK PUBL. CO.
PEARSON WR, METHODS MOL BIOL, vol. 132, 2000, pages 185 - 219
S. M. BERGE ET AL.: "describes pharmaceutically acceptable salts in detail", J. PHARMACEUTICAL SCIENCES, vol. 66, 1977, pages 1 - 19
ZIFFELS BARBARA ET AL: "Targeted delivery of calreticulin to ED-A fibronectin leads to tumor-growth retardation", JOURNAL OF BIOTECHNOLOGY, vol. 290, 2019, pages 53 - 58, XP085572667, ISSN: 0168-1656, DOI: 10.1016/J.JBIOTEC.2018.12.007 *

Similar Documents

Publication Publication Date Title
JP7535489B2 (ja) 治療用抗癌ネオエピトープワクチン
JP7181880B2 (ja) 免疫療法のためのコア／シェル構造プラットホーム
JP2022116237A (ja) ネオ抗原およびその使用方法
JP2023106599A (ja) タンパク質抗原およびその使用
JP2021527426A (ja) ネオ抗原およびその使用
TW202513579A (zh) 新抗原及其用途
EP2999485B1 (fr) Composition immunogène de peptides de la gastrine
JP2022536695A (ja) 新抗原組成物およびその使用
KR20250170533A (ko) 대상포진 mRNA 백신 및 이의 제조방법과 용도
RU2769474C2 (ru) Способы лечения рассеянного склероза с использованием аутологичных т-клеток
EP1992638B1 (fr) Peptides avant une propriete anti-tumorale et immunomodulatrice
US20200024316A1 (en) Cacna1h-derived tumor antigen polypeptide and use thereof
CN113227121A (zh) 通过特异性结合至靶细胞具有增强的多种免疫功能的嵌合抗原及其用途
JP4780540B2 (ja) サバイビン由来癌抗原ペプチド
WO2025207632A1 (fr) Compositions et méthodes pour le traitement de néoplasmes myéloprolifératifs
EP2032151A2 (fr) Procédé de traitement combiné et formulation
JP7585480B2 (ja) Ｐｄ－ｌ１の細胞外ドメインを含むキメラ抗原
US11612643B2 (en) Col14A1-derived tumor antigen polypeptide and use thereof
RU2813924C2 (ru) Неоантигены и их применение
RU2805196C2 (ru) Неоантигены и их применение
WO2023161649A1 (fr) Vaccin contre le rhinovirus
CA3274272A1 (en) HERPES ZOSTER mRNA VACCINE, PREPARATION METHOD THEREFOR, AND USE THEREOF
KR101113390B1 (ko) ＰＴＥＮ 단백질 특이적인 ｓｉＲＮＡ
HK40086688A (zh) 新抗原及其使用方法
WO2015090223A1 (fr) Protéine et son utilisation pour traiter la sclérose en plaques

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: 25720537

Country of ref document: EP

Kind code of ref document: A1