WO2025081106A2 - Compositions de vaccin à nanocorps - Google Patents

Compositions de vaccin à nanocorps Download PDF

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Publication number
WO2025081106A2
WO2025081106A2 PCT/US2024/051131 US2024051131W WO2025081106A2 WO 2025081106 A2 WO2025081106 A2 WO 2025081106A2 US 2024051131 W US2024051131 W US 2024051131W WO 2025081106 A2 WO2025081106 A2 WO 2025081106A2
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nanobody
seq
protein
based composition
domain
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WO2025081106A3 (fr
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John T. Wilson
Karan ARORA
Blaise R. KIMMEL
Hayden Michael PAGENDARM
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Vanderbilt University
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Vanderbilt University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
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    • A—HUMAN NECESSITIES
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    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6875—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin
    • A61K47/6879—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin the immunoglobulin having two or more different antigen-binding sites, e.g. bispecific or multispecific immunoglobulin
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
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    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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    • A61K2039/6031—Proteins
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    • A61K2039/6093—Synthetic polymers, e.g. polyethyleneglycol [PEG], Polymers or copolymers of (D) glutamate and (D) lysine
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    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • TECHNICAL FIELD This disclosure relates to nanobody vaccine compositions and their use in biomedical applications, such as immune therapy.
  • INTRODUCTION Improved delivery of vaccine compositions can be useful in treating many diseases, including autoimmune diseases and cancer.
  • designing vaccine compositions that can effectively modulate the innate immunity in subjects with these diseases can improve therapeutic results.
  • SUMMARY In one aspect, disclosed are protein-based compositions comprising: a nanobody domain, the nanobody domain comprising an albumin-binding nanobody, wherein the albumin- binding nanobody is capable of specifically binding albumin; and a peptide antigen domain.
  • conjugates or a pharmaceutically acceptable salt thereof, comprising: the protein-based composition as disclosed herein; a drug; a second linker attaching the peptide antigen domain at its C-terminal end to the drug.
  • pharmaceutical compositions comprising: a protein- based composition as disclosed herein or a conjugate as disclosed herein; and a pharmaceutically acceptable excipient.
  • methods of treating a disease or a disorder in a subject in need thereof comprising administering to the subject an effective amount of the protein-based composition as disclosed herein, optionally in combination with a pharmaceutically acceptable excipient.
  • methods of modulating an immune system of a subject in need thereof the method comprising administering to the subject an effective amount of the protein-based composition as disclosed herein or the conjugate as disclosed herein, optionally in combination with a pharmaceutically acceptable excipient.
  • FIG.1 is a scheme depicting the series of sortase and strain-promoted azide-alkyne cycloaddition (SPAAC) reactions used to label the C-terminus of a protein-based composition with small molecules, including Cy5, diABZI, dexamethasone, and/or synthetic peptide antigens.
  • FIG.2 shows electrospray ionization mass spectrometry (ESI-MS) spectra demonstrating nanobody conjugate purity and molecular weight.
  • ESI-MS electrospray ionization mass spectrometry
  • FIG.7 shows an SDS-PAGE gel demonstrating nanobody-Cy5 conjugate purity and molecular weight when the primary ligand contains a bicyclononyne and the secondary ligand contains an azide.
  • FIG.8 shows an SDS-PAGE gel demonstrating nanobody-antigen chemically conjugated purity and molecular weight.
  • FIG.13 is a schematic depicting the treatment timeline for prophylactic, tolerogenic nAlb-MOG 35-55 vaccination and subsequent disease challenge using an experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS).
  • EAE experimental autoimmune encephalomyelitis
  • PBS vehicle
  • P- values were determined by one-way ANOVA with post-hoc Tukey’s correction for multiple comparisons; *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001.
  • FIG.16 is a schematic depicting the treatment timeline for activating nanobody- antigen-diABZI conjugate vaccination and subsequent analysis of circulating peripheral blood mononuclear cells (PBMCs) and splenocytes.
  • FIG.17 depicts several bar graphs of the H-2Kb/SIINFEKL-Pe Tetramer + populations on Day 14 as a percentage of CD8 + T cells within the peripheral blood mononuclear cells (PBMCs) following treatment with vehicle (PBS), a full nAlb-OVA 251-270 -diABZI nanobody- antigen-drug conjugate, a mixture of the nAlb-OVA 251-270 antigen fusion and the nAlb-diABZI conjugate, a mixture of the nAlb-OVA 251-270 antigen fusion and free diABZI, and a mixture of a synthetic long peptide (SLP) variant of the SIINFEKL (SEQ ID NO:110) antigen (OVA 25
  • SLP synthetic
  • SLP synthetic long peptide
  • FIG.20 depicts several bar graphs of the central memory (T CM ), CD62L + CD44 + double-positive populations (top), and of the effector memory (T EM ), CD62L-CD44 + single- positive populations (bottom), populations on Day 21 as a percentage of Tetramer + cells within Attorney Docket No.093386-0008-WO02 the spleen following treatment with a full nAlb-OVA 251-270 -diABZI nanobody-antigen-drug conjugate, a mixture of the nAlb-OVA 251-270 antigen fusion and the nAlb-diABZI conjugate, a mixture of the nAlb-OVA 251-270 antigen fusion and free diABZI, and a mixture of a synthetic long peptide (SLP)
  • SLP synthetic long peptide
  • PBMCs peripheral blood mononuclear cells
  • FIG.23 shows tumor growth curves for mice treated with indicated formulations plotted from the day of tumor inoculation and until the death of the first mouse in each group. Statistical analyses were performed on Day 17, following the death of the first mouse in any group. P-values were determined by one-way ANOVA with post-hoc Tukey’s correction for multiple comparisons; *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001.
  • FIG.24 shows the Kaplan-Meier survival curves for mice with B16.F10-OVA tumors prophylactically treated with indicated vaccine formulations. P-values were determined via Mantel-Cox log-rank test; *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001.
  • FIG.25 is a schematic depicting the treatment timeline for C57BL/6 mice therapeutically vaccinated S.C. at the left base-of-tail with nanobody-antigen-diABZI conjugates once average B16.F10-OVA melanoma tumor volumes reached ⁇ 50-75 mm 3 .
  • FIG.26 shows the tumor growth curves for mice treated with indicated formulations plotted from the day of first treatment until the death of the first mouse in each group. Statistical analyses were performed on Day 8, following the death of the first mouse in any group. P- values were determined by one-way ANOVA with post-hoc Tukey’s correction for multiple comparisons; *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001. Attorney Docket No.093386-0008-WO02 [0038]
  • FIG.27 shows the Kaplan-Meier survival curves for mice with B16.F10-OVA tumors therapeutically treated with indicated vaccine formulations.
  • FIG.28 is a schematic of an nAlb-nPD-L1-OVA 251-270 protein fusion with a C-terminal eSrtA conjugation motif for the generation of a nAlb-nPD-L1-OVA 251-270 -diABZI protein-based composition immune checkpoint targeted cancer vaccine.
  • FIG.29 is an image of an SDS-PAGE gel demonstrating bispecific nanobody-antigen- diABZI conjugate molecular weight and purity.
  • FIG.32 shows the tumor growth curves for mice treated with indicated formulations plotted from the day of first treatment until the death of the first mouse in each group. Statistical analyses were performed on Day 10, following the death of the first mouse in any group. P- values were determined by one-way ANOVA with post-hoc Tukey’s correction for multiple comparisons; *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001.
  • FIG.33 shows the Kaplan-Meier survival curves for mice with B16.F10-OVA tumors therapeutically treated with indicated vaccine formulations.
  • each intervening number there between with the same degree of precision is explicitly contemplated.
  • the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are contemplated, and for the range 1.5-2, the numbers 1.5, 1.6, 1.7, 1.8, 1.9, and 2 are contemplated.
  • Definitions of specific functional groups and chemical terms are described in more detail below.
  • amino acid refers to naturally occurring and non-natural synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner Attorney Docket No.093386-0008-WO02 similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code.
  • Amino acids can be referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Amino acids include the side chain and polypeptide backbone portions.
  • C-terminal end refers to a fragment of a polypeptide that begins at any amino acid in the C-terminal half of the polypeptide and ends at the last amino acid of the polypeptide.
  • C-terminus refers to the last amino acid of a polypeptide.
  • drug refers to a substance that can act on a cell, virus, tissue, organ, organism, or the like, to create a change in the functioning of the cell, virus, tissue, organ, or organism. Examples of drugs include, but are not limited to, chemotherapeutics, anti- inflammatory drugs, and immunomodulating drugs.
  • a drug is capable of treating and/or ameliorating a condition or disease, or one or more symptoms thereof, in a subject.
  • Drugs of the present disclosure also include prodrug forms of the agent.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of substance are outweighed by the therapeutically beneficial effects.
  • fusion refers to a single protein or polypeptide that is produced by joining two or more originally separate genes into a single gene.
  • heterologous refers to nucleic acid comprising two or more subsequences that are not found in the same relationship to each other in nature.
  • a nucleic acid that is recombinantly produced typically has two or more sequences from unrelated genes synthetically arranged to make a new functional nucleic acid, for example, a promoter from one source and a coding region from another source. The two nucleic acids are thus heterologous to each other in this context.
  • host cell is a cell that is susceptible to transformation, transfection, transduction, conjugation, and the like with a nucleic acid construct or expression vector.
  • Host cells can be derived from plants, bacteria, yeast, fungi, insects, animals, etc.
  • the host cell includes Escherichia coli.
  • identity refers to the proportion of identical residues between a particular reference sequence and another sequence, as calculated by a pairwise alignment using the Needleman-Wunsch algorithm using a generally available alignment program, e.g., the Needle (EMBOSS) program.
  • EMBOSS Needle
  • the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of the single sequence are included in the denominator but not the numerator for the purposes of calculating identity.
  • Phage libraries can then be panned against immobilized antigens to select for nanobodies that selectively bind the antigen with high affinity.
  • the panned libraries can be used for reinfection of E. coli to obtain specific clones. Further description of nanobodies can be found in S. Muyldermans, A guide to: generation and design of nanobodies, FEBS J.2021 Apr; 288(7): 2084–2102, which is incorporated by reference herein in its entirety.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded form and complements thereof.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).
  • PNAs peptide-nucleic acids
  • N-terminal end refers to a fragment of a polypeptide that begins at the first amino acid of the polypeptide and ends at any amino acid in the N-terminal half of the polypeptide.
  • N-terminus refers to the first amino acid of a polypeptide.
  • a “peptide” or “polypeptide,” as used herein, refers to a linked sequence of two or more amino acids linked by peptide bonds.
  • the polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic.
  • Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies.
  • polypeptide refers to the amino acid sequence of a particular peptide.
  • Secondary structure refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains, Attorney Docket No.093386-0008-WO02 for example, enzymatic domains, extracellular domains, transmembrane domains, nanobody domains, and peptide antigen domains.
  • Domains are portions of a polypeptide that form a compact unit of the polypeptide and can be 8 to 1,500 amino acids in length, such as 8 to 500 amino acids in length or 8 to 50 amino acids in length.
  • Example domains include domains with enzymatic activity or ligand binding activity. Typical domains can be made up of sections of lesser organization such as stretches of beta-sheet and alpha-helices.
  • “Tertiary structure” refers to the complete three-dimensional structure of a polypeptide.
  • “Quaternary structure” refers to the three-dimensional structure formed by the noncovalent association of independent tertiary units.
  • a “motif” is a portion of a polypeptide sequence and includes at least two amino acids.
  • a motif may be 2 to 20, 2 to 15, or 2 to 10 amino acids in length. In some embodiments, a motif includes 3, 4, 5, 6, or 7 sequential amino acids.
  • a domain may be comprised of a series of the same type of motif or of different motifs.
  • “Recombinant” when used with reference to, e.g., a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed, or not expressed at all.
  • the term “specifically binds” is generally meant that a molecule (e.g., a nanobody, a protein-based composition thereof, or a conjugate thereof) binds to a target molecule when it binds to that target molecule more readily than it would bind to a random, unrelated target.
  • a molecule e.g., a nanobody, a protein-based composition thereof, or a conjugate thereof
  • nanobodies disclosed herein can specifically bind to a target molecule with nanomolar affinity. “Specific binding” does not necessarily require (although it can include) exclusive binding to a target molecule or epitope thereof.
  • the subject may be a human or a non- human.
  • the subject may be a vertebrate.
  • the subject may be a mammal.
  • the mammal may be a primate or a non-primate.
  • T cells express a T-cell receptor (TCR) on their cell surface.
  • T cell receptor (TCR) of a T cell is able to interact with immunogenic peptides (epitopes) bound to major histocompatibility complex (MHC) molecules and presented on the surface of target cells. Specific binding of the TCR triggers a signal cascade inside the T cell leading to proliferation and differentiation into a maturated effector T cell.
  • T cells may differentiate into different types of T cells. T cells may include, for example, CD8+ T cells (“killer T cells” or “cytotoxic T cells) and CD4+ T cells (“helper T cells”).
  • CD8+ T cells and CD4+ T cells may further differentiate into other types of T cells including, for example, regulatory T cells (“suppressor T cells”) and memory T cells.
  • the T cell is a memory T cell.
  • An antigen-na ⁇ ve T cell expands and differentiates into a memory T cell after encountering the cognate antigen within the context of a major histocompatibility complex (MHC) molecule on the surface of an antigen presenting cell.
  • MHC major histocompatibility complex
  • Memory T cells may be CD8+ or CD4+. Memory T cells are long-lived and can quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen.
  • Tissue-resident memory T cells are a subset of a long-lived memory T cells that occupy epithelial, mucosal, and other tissues such as skin, mucosa, lung, brain, pancreas, and gastrointestinal tract, without recirculating.
  • T RM cells may be transcriptionally, phenotypically, and functionally different from central memory (T CM ) and effector memory (T EM ) T cells that recirculate between blood, the T cell zones of secondary lymphoid organ, lymph tissues, and nonlymphoid tissues.
  • T RM cells can develop from circulating effector memory T cell precursors Attorney Docket No.093386-0008-WO02 in response to an antigen.
  • T RM cells may be CD103+.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • “Vector” as used herein means a nucleic acid sequence containing an origin of replication.
  • a vector may be capable of directing the delivery or transfer of a polynucleotide sequence to target cells, where it can be replicated or expressed.
  • a vector may contain an origin of replication, one or more regulatory elements, and/or one or more coding sequences.
  • a vector may be a viral vector, bacteriophage, bacterial artificial chromosome, plasmid, cosmid, or yeast artificial chromosome.
  • a vector may be a DNA or RNA vector.
  • a vector may be a self- replicating extrachromosomal vector.
  • Viral vectors include, but are not limited to, adenovirus vector, adeno-associated virus (AAV) vector, retrovirus vector, or lentivirus vector.
  • a vector may be an adeno-associated virus (AAV) vector.
  • the vector may encode a recombinant protein.
  • proteins and substituents thereof may be selected in accordance with permitted valence of the atoms and the substituents, such that the selections and substitutions result in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
  • Protein-Based Compositions Provided herein are protein-based compositions that can advantageously bind albumin.
  • the protein-based composition includes a nanobody domain and a peptide antigen domain.
  • the nanobody domain can include a nanobody that is capable of specifically binding albumin, where this nanobody can be referred to as an albumin-binding nanobody.
  • the ability to bind albumin can provide advantageous benefits to the protein-based composition and Attorney Docket No.093386-0008-WO02 conjugate thereof including, but not limited to, improved pharmacokinetics and pharmacodynamics.
  • the peptide antigen domain of the protein-based composition can localize to specific biological locations, such as lymph nodes, which can be beneficial for immunity/vaccine applications.
  • the nanobody domain can also include a second nanobody that is capable of specifically binding to an immune checkpoint ligand and/or an antigen presenting cell ligand. The inclusion of the second nanobody can further increase the efficacy and/or potency of the protein-based composition and conjugate thereof in e.g., immune therapy applications.
  • the protein-based composition can include the nanobody domain and the peptide antigen domain arranged in different ways.
  • the nanobody domain can be positioned N-terminal to the peptide antigen domain.
  • the protein-based composition includes, in a N-terminus to a C-terminus direction, the albumin-binding nanobody and the peptide antigen domain.
  • the albumin- binding nanobody can be N-terminal to the second nanobody
  • the peptide antigen domain can be C-terminal to the second nanobody.
  • the second nanobody can be in between the albumin-binding nanobody and the peptide antigen domain.
  • the protein-based composition includes, in a N-terminus to a C-terminus direction, the albumin- binding nanobody, the second nanobody, and the peptide antigen domain.
  • the nanobody domain can be attached (e.g., covalently) to the peptide antigen domain through a first linker.
  • the nanobody domain can be attached at its C-terminal end to the N-terminal end of the peptide antigen domain through the first linker.
  • the first linker can include a peptide linker, a sortase moiety, or a combination thereof.
  • the first linker can also be one that is formed from biorthogonal chemistry.
  • the peptide antigen domain is flanked on either side by a first linker, such as a peptide linker.
  • a first linker such as a peptide linker.
  • the peptide linker can provide flexibility between the different domains.
  • the peptide linker can also be cleavable.
  • the peptide linker can be cleavable by cathepsins and/or proteasomes.
  • Example peptide linkers include, but are not limited to, GGGS (SEQ ID NO:99), SGSETPGTSESA (SEQ ID NO:100), SLVR (SEQ ID NO:101), SLVRYLL (SEQ ID NO:102), valine–citrulline linkers, and alanine-based linkers (e.g., dialanine AA).
  • the peptide linker is 2 to 30 amino acids in length, such as 2 to 25, 3 to 30, 4 to 25, or 2 to 20.
  • the sortase moiety can include a sortase recognition site.
  • the sortase moiety can include an amino acid sequence of LPXT (SEQ ID NO:1) (e.g., a sortase recognition site), wherein X is any amino acid.
  • the sortase moiety includes an amino Attorney Docket No.093386-0008-WO02 acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.
  • the sortase moiety and/or amino acid sequence thereof is described as part of a sequence encoding a nanobody.
  • the sortase moiety may also include a hydrophilic moiety, such as polyethylene glycol as described herein.
  • the protein-based composition is made can dictate the type of linker used to attach the nanobody domain to the peptide antigen domain.
  • the nanobody domain and the peptide antigen domain are made as a fusion protein.
  • the nanobody domain and the peptide antigen domain can be expressed within the same protein.
  • the nanobody domain can be attached to the peptide antigen domain by a first linker that includes a peptide linker as described herein.
  • the albumin-binding nanobody is attached to the peptide antigen domain by a first linker that includes a peptide linker as described herein.
  • the nanobody domain and the peptide antigen domain can be attached by chemical methods.
  • the nanobody domain or nanobody therein can be designed to include a sortase recognition site.
  • An example sortase recognition site is LPXT (SEQ ID NO:1), wherein X is any amino acid.
  • the sortase recognition site includes an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.
  • the sortase recognition site can be at the C-terminus of the nanobody domain and can allow for site-specific conjugation of a primary amine reagent via sortase.
  • the primary amine reagent can include a first reactive group.
  • the reactive amine reagent is of formula (I): (I), wherein: X 1 is the first reactive group (e.g., BCN) and n’ is 2 to 20.
  • n’ is 2 to 4, 2 to 6, 2 to 10, 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, or 10 to 20.
  • n’ is greater than 2, greater than 3, greater than 4, greater than 5, greater than 6, greater than 7, greater than 8, greater than 9, or greater than 10.
  • the second nanobody is capable of specifically binding to an immune checkpoint ligand.
  • An “immune checkpoint ligand” refers to ligand-receptor pairs expressed on immune cells that inhibit or stimulate the immune response.
  • the immune checkpoint ligand can be present on a cell surface.
  • Example immune checkpoint ligands include, but are not limited to, CTLA-4, PD-1, PD-L1, B7-H3, B7-H4, HVEM, GITRL, CD80/86, CD155, PD-L2, Galectin 9, LAG3, TIM3, VISTA, TIGIT, PD1, MMR, and GITR.
  • the second nanobody can have a K d to an antigen presenting cell ligand as described above for the albumin-binding nanobody to albumin.
  • the second nanobody includes an amino acid sequence selected from the group consisting of SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:106, SEQ ID NO:107, and SEQ ID NO:108.
  • the second nanobody includes an amino acid sequence of SEQ ID NO:94.
  • the first nanobody can be attached to the second nanobody through a peptide linker.
  • the peptide linker attaching the first and the second nanobody can be any of the peptide linkers described for the first linker (e.g., between the nanobody domain and the peptide antigen domain).
  • the peptide antigen domain is flanked on either side by a peptide linker.
  • the nanobody domain includes an albumin-binding nanobody that is capable of specifically binding to albumin and a second nanobody that is capable of specifically binding to an immune checkpoint ligand.
  • the nanobody domain includes an albumin-binding nanobody including an amino acid sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 81, and a second nanobody including an amino acid sequence selected from the group consisting of SEQ ID NO:94 and SEQ ID NO:95.
  • the nanobody domain includes an amino acid sequence selected from the group consisting of SEQ ID NO:80, SEQ ID NO: 94, and a combination thereof.
  • Attorney Docket No.093386-0008-WO02 [0096]
  • the nanobody domain includes an albumin-binding nanobody that is capable of specifically binding to albumin and a second nanobody that is capable of specifically binding to an antigen presenting cell ligand.
  • the nanobody domain includes an albumin-binding nanobody including an amino acid sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 81, and a second nanobody including an amino acid sequence selected from the group consisting of SEQ ID NO:106 to SEQ ID NO:108.
  • the nanobody domain includes an amino acid sequence selected from the group consisting of SEQ ID NO:80, SEQ ID NO:106 to SEQ ID NO:108, and a combination thereof.
  • the nanobody domain includes an amino acid sequence of SEQ ID NO:5 to SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:88, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:106 to SEQ ID NO:108, or a combination thereof.
  • the nanobodies disclosed herein can be commercially purchased or provided by recombinant expression.
  • the peptide antigen domain includes a peptide antigen.
  • a “peptide antigen” refers to a peptide capable of being bound by an antibody or a T cell receptor.
  • the term “peptide antigen” also encompasses T-cell epitopes.
  • a peptide antigen is additionally capable of being recognized by the immune system and/or being capable of inducing a humoral immune response and/or cellular immune response leading to the activation of B-lymphocytes and/or T-lymphocytes.
  • the peptide antigen contains or is linked to a Th cell epitope.
  • a peptide antigen can have one or more epitopes (B-epitopes and T-epitopes). Accordingly, the peptide antigen can facilitate modulating an immune response in a subject.
  • the peptide antigen domain consists of a peptide antigen.
  • the peptide antigen can have a molecular weight of about 1 kiloDalton (kDa) to about 10 kDa, such as about 2 kDa to about 9 kDa, about 3 kDa to about 8 kDa, about 1 kDa to about 5 kDa, about 3 kDa to about 10 kDa, or about 5 kDa to about 10kDa.
  • the peptide antigen has a molecular weight of greater than 1 kDa, greater than 2 kDa, greater than 3 kDa, or greater than 4 kDa.
  • the peptide antigen domain includes an amino acid sequence of SEQ ID NO:96 to SEQ ID NO:98.
  • the protein-based composition includes, in a N-terminus to a C-terminus direction: the nanobody domain, the nanobody domain comprising the albumin- binding nanobody, wherein the albumin-binding nanobody is capable of specifically binding albumin, and the second nanobody, wherein the second nanobody is capable of specifically binding to an immune checkpoint ligand; and the peptide antigen domain, the peptide antigen domain comprising a tumor antigen.
  • the protein-based composition includes, in a N-terminus to a C-terminus direction: the nanobody domain, the nanobody domain comprising the albumin- binding nanobody, wherein the albumin-binding nanobody is capable of specifically binding albumin, and the second nanobody, wherein the second nanobody is capable of specifically binding to an antigen presenting cell ligand; and the peptide antigen domain, the peptide antigen domain comprising a tumor antigen.
  • D. Polynucleotides [00112] Further provided are polynucleotides encoding the protein-based compositions detailed herein, including the nanobody domain, the albumin-binding nanobody, the second nanobody, and the peptide antigen domain.
  • a vector may include the polynucleotide encoding the protein-based composition or domains thereof detailed herein.
  • To obtain expression of a polypeptide one may subclone the polynucleotide encoding the polypeptide into an expression vector that contains a promoter to direct transcription, a transcription/translation terminator, and if for a nucleic acid encoding a protein, a ribosome binding site for translational initiation.
  • An example of a vector is pet24. Suitable bacterial promoters are well known in the art.
  • a host cell transformed or transfected with an expression vector comprising a polynucleotide encoding the protein-based composition or domains thereof as detailed herein.
  • the conjugate can include the protein-based composition as disclosed herein and a drug attached to the protein-based composition by a second linker. Because the protein-based Attorney Docket No.093386-0008-WO02 composition is able to bind to albumin, the conjugate thereof can also specifically bind to albumin. The ability to bind albumin can provide advantageous benefits to the conjugate including, but not limited to, improved pharmacokinetics and pharmacodynamics of the attached drug. [00114]
  • the disclosed conjugates may exist as salts, such as pharmaceutically acceptable salts.
  • pharmaceutically acceptable salt refers to salts or zwitterions of the conjugates which are water or oil-soluble or dispersible, suitable for administration to a subject (e.g., treatment of disorders) without undue toxicity, irritation, and allergic response, commensurate with a reasonable benefit/risk ratio and effective for their intended use.
  • the salts may be prepared during the final isolation and purification of the conjugates or separately by reacting an amino group of the conjugates with a suitable acid.
  • the conjugate may be dissolved in a suitable solvent and treated with at least one equivalent of an acid, like hydrochloric acid.
  • the resulting salt may precipitate out and be isolated by filtration and dried under reduced pressure.
  • salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, isethionate, fumarate, lactate, maleate, methanesulfonate, naphthylenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, oxalate, maleate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, glutamate, para-toluenesulfonate, undecanoate, hydrochloric
  • Amino groups of the conjugates may also be quaternized with alkyl chlorides, bromides and iodides such as methyl, ethyl, propyl, isopropyl, butyl, lauryl, myristyl, stearyl and the like.
  • Basic addition salts may be prepared during the final isolation and purification of the disclosed conjugates by reaction of a carboxyl group with a suitable base such as the hydroxide, carbonate, or bicarbonate of a metal cation such as lithium, sodium, potassium, calcium, magnesium, or aluminum, or an organic primary, secondary, or tertiary amine.
  • Quaternary amine salts can be prepared, such as those derived from methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine and N,N’-dibenzylethylenediamine, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like.
  • the conjugate includes a linker attaching (e.g., covalently) the protein-based composition to the drug, which can be referred to as a second linker.
  • the second linker can attach the protein-based composition to the drug through the peptide antigen domain, e.g., at its C-terminal to the drug.
  • the second linker can include a sortase moiety.
  • the sortase moiety can be attached to the peptide antigen domain. In some embodiments, the sortase moiety is located at the C-terminus of the peptide antigen domain.
  • the sortase moiety can include a sortase recognition site.
  • the sortase moiety can include an amino acid sequence of LPXT (SEQ ID NO:1), wherein X is any amino acid.
  • the sortase moiety includes an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.
  • the sortase moiety and/or amino acid sequence thereof is described as part of a sequence encoding a protein-based compositions or domains thereof.
  • the sortase moiety may also include a hydrophilic moiety, such as polyethylene glycol as disclosed herein.
  • the second linker can also include a hydrophilic moiety.
  • the hydrophilic moiety can attach the sortase moiety to the drug. Due to the hydrophobicity of the drug, such as diABZI, the second linker can advantageously aid in solubilizing the drug.
  • An example hydrophilic linker includes a hydrophilic polymer such as polyethylene glycol (PEG).
  • the second linker includes a PEG moiety that, e.g., attaches the sortase moiety to the drug.
  • the PEG moiety can include a varying number of ethylene glycol repeats.
  • the PEG moiety can include 3 to 20 ethylene glycol repeats, such as 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, or 10 to 20.
  • the PEG moiety includes greater than 2 ethylene glycol repeats, greater than 3 ethylene glycol repeats, greater than 4 ethylene glycol repeats, greater than 5 ethylene glycol repeats, greater than 6 ethylene glycol repeats, greater than 7 ethylene glycol repeats, greater than 8 ethylene glycol repeats, greater than 9 ethylene glycol repeats, or greater than 10 ethylene glycol repeats.
  • the PEG moiety is of formula (a): wherein: n is 2 to 20. In formula (a) as depicted, the drug would be attached to the right side of the PEG moiety of formula (a). Further description of how the second linker and the PEG moiety are attached can be found herein and in the Examples.
  • compositions including the Protein-Based Composition and/or Conjugate Thereof
  • compositions that include the protein-based composition and/or conjugate thereof and a pharmaceutically acceptable excipient, where such compositions can also be referred to as a pharmaceutical composition.
  • the description of the protein-based composition, nanobody domain, peptide antigen domain, drug, conjugate, and linkers above can be applied to the disclosed compositions.
  • pharmaceutically acceptable excipient means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • materials which can serve as pharmaceutically acceptable excipients are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, corn starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic s
  • the route by which the composition is administered, and the form of the composition can dictate the type of excipient to be used.
  • the pharmaceutically acceptable excipient includes buffering agents (e.g., phosphate buffered saline), carbohydrates (e.g., glucose, trehalose, starch, etc.) solubilizers, solvents, antimicrobial preservatives, antioxidants, suspension agents, or a combination thereof.
  • buffering agents e.g., phosphate buffered saline
  • carbohydrates e.g., glucose, trehalose, starch, etc.
  • solubilizers e.g., solubilizers, solvents, antimicrobial preservatives, antioxidants, suspension agents, or a combination thereof.
  • solvents e.g., glucose, trehalose, starch, etc.
  • the protein-based composition, conjugate thereof, and composition thereof may be suitable for administration to a subject (such as a patient, which may be a human or non- human) well known to those skilled in the pharmaceutical art.
  • the protein-based composition, conjugate thereof, and composition thereof may be prepared for administration to a subject.
  • Such protein-based composition, conjugate thereof, and composition thereof can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular subject, and the route of administration.
  • the protein-based composition, conjugate thereof, and composition thereof can be administered prophylactically or therapeutically.
  • the protein-based composition, conjugate thereof, and composition thereof can be administered in an Attorney Docket No.093386-0008-WO02 amount sufficient to induce a response.
  • the protein-based composition, conjugate thereof, and composition thereof can be administered to a subject in need thereof in an amount sufficient to elicit a therapeutic effect.
  • An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition of the regimen administered, the manner of administration, the stage and severity of the disease, the general state of health of the patient, and the judgment of the prescribing physician.
  • the protein-based composition, conjugate thereof, and composition thereof can be administered via a variety of routes.
  • Typical delivery routes include parenteral administration, e.g., intradermal, intramuscular or subcutaneous delivery.
  • Other routes include oral administration, intranasal, intravaginal, transdermal, intravenous, intraarterial, intratumoral, intraperitoneal, and epidermal routes.
  • the protein-based composition, conjugate thereof, or composition thereof is administered intravenously, subcutaneously, intradermally, intramuscularly, or intraperitoneally.
  • protein-based composition, conjugate thereof, or composition thereof is administered intravenously or subcutaneously.
  • the protein-based composition and the conjugate thereof can act as a vaccine, they can modulate the subject’s immune system in any way that a vaccine can traditionally modulate an immune system.
  • Example ways that the protein-based composition and conjugate thereof can modulate an immune system include, but are not limited to, increasing an amount of regulatory T cells in the subject specific to the peptide antigen domain, increasing an amount of CD8+ T cells (which includes cytotoxic T cells (CTLs)) in the subject, increasing the amount of CD4+ Th cells, or a combination thereof.
  • TTLs cytotoxic T cells
  • Th cells include, but are not limited to, CD4+ Th1, Th17 cells, CD4+ Th2 cells, and CD4+ Th9 cells.
  • Example 1 Cell Lines and Materials. All chemicals involved in synthesis of target compounds were reagent grade unless stated otherwise. DNase, isopropyl thiogalactoside (IPTG), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich. Azido-PEG 3 -Amine and DBCO- PEG 12 -NHS Ester were purchased from Broadpharm. Magnesium sulfate, sodium hydroxide, sodium azide, sodium acetate, sodium azide, sodium chloride, sodium bicarbonate, sodium hydroxide, 2xYT media, kanamycin, Nickel NTA resin, and all other organic solvents were purchased from Thermo Fisher Scientific.
  • the construct was transformed into chemically competent DH5 ⁇ E. coli and plated on LB agar with Kanamycin.
  • the sequence verified nanobody was transformed in pET28b into T7 Shuffle Express (New England Biolabs) with E. coli as the expression strain. [00161] Sequence details. Attorney Docket No.093386-0008-WO02 Engineered Sortase A (eSrtA) Sequence (No Start Codon).
  • nGFP-OVA 251-270 – Ligation Tag MQVQLQESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSS YEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS – GGGSGLEQLESIINFEKLTEWTSSGGGS – LPETGGHHHHHHEPEA (SEQ ID NO:86) Formula: C 758 H 1152 N 222 O 246 S 6 Attorney Docket No.093386-0008-WO02 Molecular Weight: 17503.19 Da ⁇ 280 32555 M -1 cm -1 Anti-GFP-MOG 35-55 (nGFP-MOG 35-55 ) Sequence (No Start Codon).
  • the culture was incubated at 30 oC, with shaking at 250 RPM, for 16 hours. Each culture was transferred to a 2 L baffled flask containing 500 mL of 2xYT media and 500 ⁇ L of Kanamycin (25 mg) and shaken at 30 oC in an Innova 42R (New Brunswick Scientific) incubator for 4.5-5 hours (until the OD600 reached ⁇ 0.8). The cultures were then induced with IPTG (2.5 mM final concentration). The induced cultures were shaken overnight (20-24 hours) at 30 oC. The bacteria were harvested the next day by centrifugation (3900 rpm for 10 min) and the pellet was reconstituted in 1x PBS with Dnase with a tablet of protease inhibitor cocktail (EDTA free).
  • EDTA free protease inhibitor cocktail
  • the cells were lysed by sonication on an ice bath in 5 second increments over 10 minutes.
  • the resulting bacterial lysate was centrifuged (11000 rpm for 20 min) to remove cellular debris.
  • the lysate was added to a 50 mL Kontes Flex column (Kimbal Kontes Glassware) containing 3 mL of Nickel NTA histidine binding resin that was preequilibrated with 1x PBS buffer. This column was placed on a rotating shaker at room temperature for 1-2 hrs. After this period, the supernatant was drained from the column using gravity and the column washed with 1x PBS buffer twice.
  • the eluate was then concentrated to 0.5 mL in a 15 mL Microcon 10 kDa Centrifugal Filter Unit (Millipore) and subsequently purified by size exclusion chromatography (SEC) via an Akta FPLC (Cytiva), on a Hi-Load 16/60 Superdex 200 column using 1x PBS and 0.02% NaN3, pH 7.4 at 4 oC as the running buffer. Pure fractions were determined by SDS-PAGE, pooled together with buffer exchange to 1x PBS not containing NaN 3 , and stocked at either -20 oC or 4 oC. [00163] Enzymatic Bioconjugation and Click Chemistry Reactions.
  • Bioconjugation reactions occurred in mild conditions (20 mM HEPES at pH 7.4, 150 mM NaCl, and 10 mM CaCl 2 ) Attorney Docket No.093386-0008-WO02 between eSrtA (100 ⁇ M) and a nanobody containing a C-terminal ligation tag (75 ⁇ M) using a primary amine containing functional group (20 mM). Reactions occurred with mixing by a rotary shaker overnight (16 h) and were quenched by the addition of a 1:1 volume of a chelating agent EDTA containing solution (20 mM HEPES at pH 7.4, 300 mM NaCl, and 10 mM EDTA) for one hour.
  • a chelating agent EDTA containing solution (20 mM HEPES at pH 7.4, 300 mM NaCl, and 10 mM EDTA
  • ESI-MS data were collected using an Agilent 6210A time-of-flight (TOF) mass spectrometer at a range of 50-20,000 m/z over a period of two minutes. Data were analyzed with Agilent MassHunter IM-MS Acquisition Data software to reveal m/z data, where files were condensed across the two-minute run. These m/z data were deconvoluted using a maximum entropy deconvolution calculation using UniDec to give the deconvoluted mass spectra using background subtraction between a range of 1,000-5,000 m/z and with an export range of 5,000-50,000 Da. [00166] Synthesis and NMR Verification of DBCO-PEG 12 -diABZI.
  • N 3 -GGGS-GLEQLESIINFEKLTEWTSS N 3 -OVA 251-270
  • N 3 -GGGS- ISQAVHAAHAEINEAGR N 3 -OVA 323-339
  • N 3 -GGGS- MEVGWYRSPFSRVVHLYRNGK N 3 -MOG 35-55
  • CEM Corporation The peptide antigens N 3 -GGGS-GLEQLESIINFEKLTEWTSS (N 3 -OVA 251-270 ) (SEQ ID NO:103), N 3 -GGGS- ISQAVHAAHAEINEAGR (N 3 -OVA 323-339 ) (SEQ ID NO:104), and N 3 -GGGS- MEVGWYRSPFSRVVHLYRNGK (N 3 -MOG 35-55 ) (SEQ ID NO:105) were synthesized using a LibertyBlue 2.0 automated microwave peptide synthesizer (CEM Corporation). A polystyrene resin pre-loaded with the C
  • azido acetic acid N 3 -CH 2 -COOH
  • DIC/Oxyma coupling to generate a reactive azide at the N-terminus of the peptide.
  • the peptide was then cleaved from the resin using a cleavage cocktail of 92.5% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIPS), 2.5% dodecanethiol (DODT), and 2.5% DI H 2 O for 3 h at RT.
  • TFA trifluoroacetic acid
  • TIPS triisopropylsilane
  • DODT dodecanethiol
  • the cleaved peptide was then precipitated in cold diethyl ether, dried under vacuum. Dried crude peptide was redissolved in H 2 O and/or acetonitrile (AN), purified via semi-preparative high performance liquid chromatography (HPLC) (Waters) using an H 2 O:AN gradient and a C18 column. Collected fractions were pooled, and AN was removed using a rotary evaporator. The water-dissolved peptide was flash frozen, and water was removed via lyophilization (Labconco). The lyophilized peptide was characterized using analytical HPLC and LC-MS and stored at -20 °C.
  • HPLC high performance liquid chromatography
  • albumin- binding molecules While several albumin- binding molecules have been described, the experimental platform described below was built from a nanobody with high affinity for albumin because nanobodies are highly modular and programmable via genetic engineering, are molecularly well-defined, are amenable to scalable industrial manufacturing, and are components of approved and clinically advanced therapeutics, including ozoralizumab, which contains an anti-albumin nanobody domain.
  • a nanobody domain — termed nAlb – that binds with nanomolar affinity to serum albumin was recombinantly expressed.
  • nAlb – that binds with nanomolar affinity to serum albumin was recombinantly expressed.
  • a peptide antigen flanked by two flexible spacers (GGGS) (SEQ ID NO:99) was cloned to generate an nAlb-antigen fusion.
  • GGGS flexible spacers
  • a variant of the STING agonist diABZI was synthesized that was functionalized with an azide-reactive DBCO group and a PEG 12 spacer (DBCO-PEG 12 -diABZI). Conjugation of the NH 2 -PEG 3 -N 3 , sulfo-Cy5-DBCO, and/or DBCO-PEG 12 -diABZI was confirmed via SDS PAGE and/or ESI-MS (FIG.2, FIG.3, FIG. 4, and FIG.5). A list of antigens is provided in Table 5. Table 5. Antigens incorporated into clonally expressed nAlb-antigen fusion vaccines.
  • nAlb albumin-targeting nanobody
  • the nAlb nanobody was designed to include the eSrtA selective ligation tag (LPETGGHHHHHHEPEA) (SEQ ID NO:4) at the C-terminus to facilitate the site-specific conjugation of a primary amine containing small molecule following the LPET motif. It was demonstrated that this approach could be used to ligate the azide reactive endo-BCN-PEG 3 -NH 2 to the C-terminus of nAlb.
  • LETGGHHHHHHEPEA eSrtA selective ligation tag
  • mice were administered 2 nmol of nAlb-Cy5 or nGFP-Cy5 S.C. at the left base-of-tail.
  • Plasma Cy5 concentrations were fit to a two-compartment model of S.C. injection, and calculated serum half-lives (t 1/2 ), absorption constants (k abs ), elimination constants (k clear ), and areas under the curves (AUC) are provided in Table 7. Fusion to nAlb enhanced peptide antigen half-life, showed both its absorption into and elimination from circulation, and enhanced the absolute bioavailability of the peptide antigen. Table 7. Calculated pharmacokinetic parameters of Cy5 conjugated nanobodies.
  • the average radiant efficiency of the Cy5 signal per unit area of tissue was calculated and indicated that conjugation to nAlb significantly increased Cy5 accumulation in all organs collected (FIG.10), most notably in the iLN (FIG.11), as lymphoid tissues are the major sites of T cell priming by antigen presenting cells (APCs) – a necessary process during the elicitation of a vaccine response.
  • APCs antigen presenting cells
  • lymphoid tissues including the spleen, draining iLN, and irrelevant axillary lymph node (aLN) were processed and stained for flow cytometry.
  • APC phagocytic antigen-presenting cell
  • nAlb-OVA 251-270 peptide vaccine can only induce CD8 + T cell responses.
  • Therapeutic vaccination with nAlb-OVA 251-270 /nAlb-diABZI slows B16.F10-OVA melanoma growth and extends mouse survival. Finally, the efficacy of nAlb-OVA 251-270 /nAlb- diABZI vaccination was tested in a therapeutic model of B16.F10-OVA melanoma.
  • mice were inoculated with 5 x 10 5 B16.F10-OVA melanoma cells S.C. on the right flank. Tumors were measured every other day, and treatments began on Day 0 when the average tumor volume reached approximately 50-75 mm 3 .
  • Mice were administered 1.171 nmol OVA 251-270 , either fused to nAlb or as a free peptide, and 1.25 ⁇ g diABZI, either fused to nAlb or as a free drug, S.C. on Days 0, 4, and 8.
  • coli chemically competent cells were purchased from New England Biolabs (NEB).
  • Qiaprep Miniprep Spin kits were purchased from Qiagen.
  • THP1-Dual and A549-Dual cell lines were purchased from InvivoGen.
  • the murine melanoma cell line B16.F10-OVA was cultured in DMEM supplemented with 2 mM L-glutamine, 4.5 g/L glucose, 10% HI-FBS, and 100 U*ml -1 penicillin/100 ⁇ g*mL -1 streptomycin.
  • Ovalbumin (OVA) expression was maintained through continuous selection using Geneticin (G418; Gibco) after every cell passage at a concentration of 380 ⁇ g*mL -1 .
  • nPD-L1-OVA 251-270 – Ligation Tag MQVQLQESGGGLVHPGGSLRLSCATSGSIFSIISMGWYRQAPGKQRELVALVFRGGSTVYAD SVKGRFTISGDIAKSTVYLQMDSLKPEDTAVYYCNAKPIGTAQYWGQGTQVTVSS – GGGSGLEQLESIINFEKLTEWTSSGGGS – LPETGGHHHHHHEPEA (SEQ ID NO:91) Formula: C 752 H 1163 N 211 O 238 S 5 Molecular Weight: 17128.08 Da ⁇ 280 25565 M -1 cm -1 Anti-GFP – Anti-PD-L1 (GP) Sequence (No Start Codon).
  • the culture was incubated at 30 oC, with shaking at 250 RPM, for 16 hours. Each culture was transferred to a 2 L baffled flask containing 500 mL of 2xYT media and 500 ⁇ L of Kanamycin (25 mg) and shaken at 30 oC in an Innova 42R (New Brunswick Scientific) incubator for 4.5-5 hours (until the OD600 reached ⁇ 0.8). The cultures were then induced with IPTG (2.5 mM final concentration). The induced cultures were shaken overnight (20-24 hours) at 30 oC. The bacteria were harvested the next day by centrifugation (3900 rpm for 10 min) and the pellet was reconstituted in 1x PBS with Dnase with a tablet of protease inhibitor cocktail (EDTA free).
  • EDTA free protease inhibitor cocktail
  • the cells were lysed by sonication on an ice bath in 5 second increments over 10 minutes.
  • the resulting bacterial lysate was centrifuged (11000 rpm for 20 min) to remove cellular debris.
  • the lysate was added to a 50 mL Kontes Flex column (Kimbal Kontes Glassware) containing 3 mL of Nickel NTA histidine binding resin that was preequilibrated with 1x PBS buffer. This column was placed on a rotating shaker at room temperature for 1-2 hrs. After this period, the supernatant was drained from the column using gravity and the column washed with 1x PBS buffer twice.
  • the eluate was then concentrated to 0.5 mL in a 15 mL Microcon 10 kDa Centrifugal Filter Unit (Millipore) and subsequently purified by size exclusion chromatography (SEC) via an Akta FPLC (Cytiva), on a Hi-Load 16/60 Superdex 200 column using 1x PBS and 0.02% NaN 3 , pH 7.4 at 4 oC as the running buffer. Pure fractions were determined by SDS-PAGE, pooled together with buffer exchange to 1x PBS not containing NaN 3 , and stocked at either -20 oC or 4 oC. [00187] Enzymatic Bioconjugation and Click Chemistry Reactions.
  • Bioconjugation reactions occurred in mild conditions (20 mM HEPES at pH 7.4, 150 mM NaCl, and 10 mM CaCl 2 ) between eSrtA (100 ⁇ M) and a nanobody containing a C-terminal ligation tag (75 ⁇ M) using a primary amine containing functional group (20 mM). Reactions occurred with mixing by a rotary shaker overnight (16 h) and were quenched by the addition of a 1:1 volume of a chelating agent EDTA containing solution (20 mM HEPES at pH 7.4, 300 mM NaCl, and 10 mM EDTA) for one hour.
  • a chelating agent EDTA containing solution (20 mM HEPES at pH 7.4, 300 mM NaCl, and 10 mM EDTA
  • ESI-MS data were collected using an Agilent 6210A time-of-flight (TOF) mass spectrometer at a range of 50-20,000 m/z over a period of two minutes. Data were analyzed with Agilent MassHunter IM-MS Acquisition Data software to reveal m/z data, where files were condensed across the two-minute run. These m/z data were deconvoluted using a maximum entropy deconvolution calculation using UniDec to give the deconvoluted mass spectra using background subtraction between a range of 1,000-5,000 m/z and with an export range of 5,000-50,000 Da. [00190] Synthesis and NMR Verification of DBCO-PEG 12 -diABZI.
  • a STING agonist was generated that features a reactive amine handle, which was synthesized in four steps. Briefly, aryl amination of an aryl chloride 1 with an amine 2 gave a dinitro analog, compound 3. The di- nitro compound 3 was subjected to reduction using sodium dithionite in methanol, generating a di-amine moiety 4. Compound 4 was then treated with isothiocyanate, followed by EDC coupling, to reveal a boc-protected analog, compound 5. Next, the boc-group from compound 5 was deprotected by treating with TFA:DCM.
  • PD-L1 is highly expressed by immunosuppressive APCs in the TME.
  • TAMs tumor associated macrophages
  • TAMs tumor associated macrophages
  • Activating these APCs, in particular the tumor associated macrophages (TAMs) can be critical for the generation of a “hot” inflamed microenvironment as activated TAMs secrete many of the necessary cytokines and chemokines required to initiate potent antitumoral CD8 + T cell infiltration into the TME. Additionally, many of these TAMs traffic to lymphoid tissues, where this vaccine can also localize, where they can present peptide antigen to prime CD8 + T cells. [00197] 4)
  • the inherent blockade of the PD-L1/PD-1 immune checkpoint induced by a PD-L1 targeting nanobody vaccine can mimic the effects of anti-PD-L1 ICB therapy.
  • nAlb was recombinantly expressed at the N-terminus of the fusion protein.
  • a XTEN linker (SGSETPGTSESA) (SEQ ID NO:100) was introduced, followed by nPD-L1.
  • GGGS flexible spacers
  • a selective ligation tag (LPETGGHHHHHHEPEA) (SEQ ID NO:4) was cloned to include a site-specific substrate for an enzymatic engineered sortase pentamutant A (eSrtA) mediated ligation of any primary amine containing small molecule to the C-terminal of the nAlb-antigen fusion (Fig.27).
  • nAlb-nPD-L1-OVA 251-270 -diABZI inhibits cancer progression and extends mouse survival in a B16.F10-OVA model of melanoma.
  • Female 6–8-week C57BL/6 mice were inoculated with 5 x 10 5 B16.F10-OVA melanoma cells S.C. in the right flank.
  • Tumors were measured every other day, and treatments began on Day 0 when the average tumor volume reached ⁇ 50-75 mm 3 .
  • Mice were administered 1.25 ⁇ g diABZI via S.C. injection, an equivalent 1.47 nmol nPD-L1 and/or peptide antigen via S.C. injection, and/or 100 ⁇ g aPD-L1 ICB via I.P. injection on Days 0, 4, and 8 (FIG.30).
  • all treatment groups except for aPD-L1 ICB alone significantly slowed tumor growth and extended survival, however none to the equivalent degree as AP-OVA 251-270 (FIG.31, FIG.32, and FIG.33).
  • a protein-based composition comprising: a nanobody domain, the nanobody domain comprising an albumin-binding nanobody, wherein the albumin-binding nanobody is capable of specifically binding albumin; and a peptide antigen domain.
  • the albumin-binding nanobody has a binding affinity (K d ) to albumin of less than or equal to 100 nM at a pH of about 7 to about 8.
  • the albumin- binding nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 81.
  • Attorney Docket No.093386-0008-WO02 [00205] Clause 4.
  • the immune checkpoint ligand is CTLA-4, PD-1, PD-L1, B7-H3, B7-H4, HVEM, GITRL, CD80/86, CD155, PD-L2, Galectin 9, LAG3, TIM3, VISTA, TIGIT, PD1, MMR, or GITR.
  • the second nanobody comprises an amino acid sequence of SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:106, SEQ ID NO:107, or SEQ ID NO:108.
  • Clause 9. The protein-based composition of any one of clauses 1-8, wherein the peptide antigen domain comprises a tumor antigen.
  • Clause 10. The protein-based composition of any one of clauses 1-9, wherein the peptide antigen domain comprises an amino acid sequence of SEQ ID NO:96, SEQ ID NO:97, or SEQ ID NO:98. [00212] Clause 11.
  • the protein-based composition of any one of clause 4-12 comprising, in a N-terminus to a C-terminus direction: the nanobody domain, the nanobody domain comprising the albumin-binding nanobody, wherein the albumin-binding nanobody is capable of specifically binding albumin, and the second nanobody, wherein the second nanobody is capable of specifically binding to an immune checkpoint ligand; and the peptide antigen domain, the peptide antigen domain comprising a tumor antigen.
  • Clause 16 The protein-based composition of clause 14 or 15, wherein the peptide linker comprises an amino acid sequence of GGGS (SEQ ID NO:99), SGSETPGTSESA (SEQ ID NO:100), SLVR (SEQ ID NO: 101), SLVRYLL (SEQ ID NO:102), or a combination thereof.
  • Clause 17 The protein-based composition of any one of clauses 1-16 comprising an amino acid sequence that is at least 95% identical to any one of SEQ ID NO:84 to SEQ ID NO:87, SEQ ID NO:89 to SEQ ID NO:91, SEQ ID NO:93, or SEQ ID NO:109.
  • a conjugate, or a pharmaceutically acceptable salt thereof comprising: the protein-based composition of any one of clauses 1-17; a drug; a second linker attaching the peptide antigen domain at its C-terminal end to the drug.
  • Clause 19 The conjugate of clause 18, or a pharmaceutically acceptable salt thereof, wherein the second linker comprises a sortase moiety, the sortase moiety comprising an amino acid sequence of LPXT (SEQ ID NO:1), wherein X is any amino acid.
  • Clause 20 The conjugate of clause 19, or a pharmaceutically acceptable salt thereof, wherein the second linker comprises a polyethylene glycol (PEG) moiety attaching the sortase moiety to the drug.
  • PEG polyethylene glycol
  • Clause 21 The conjugate of clause 20, or a pharmaceutically acceptable salt thereof, wherein the PEG moiety is of formula (a): wherein: n is 2 to 20.
  • Clause 22 The conjugate of any one of clauses 18-21, or a pharmaceutically acceptable salt thereof, wherein the drug comprises an immunomodulator, an agonist, an antagonist, an inhibitor, or a hormone.
  • Clause 23 The conjugate of any one of clauses 18-22, or a pharmaceutically acceptable salt thereof, wherein the immunomodulator comprises a STING agonist.
  • nAlb-OVA 251-270 – Ligation Tag Attorney Docket No.093386-0008-WO02 MEVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS – GGGSGLEQLESIINFEKLTEWTSSGGGS – LPETGGHHHHHHEPEA (SEQ ID NO:84) Anti-Albumin-MOG 35-55 (nAlb-MOG 35-55 ) Sequence (No Start Codon).
  • nGFP-OVA 251-270 Ligation Tag MQVQLQESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSS YEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS – GGGSGLEQLESIINFEKLTEWTSSGGGS – LPETGGHHHHHHEPEA (SEQ ID NO:90) Attorney Docket No.093386-0008-WO02 Anti-PD-L1-OVA 251-270 (nPD-L1-OVA 251-270 ) Sequence (No Start Codon).
  • Molecular Weight: 14763.30 Da ⁇ 280 21095 M -1 cm -1

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Abstract

L'invention concerne des compositions à base de protéines et des conjugués de celles-ci qui peuvent tirer profit de propriétés d'administration bénéfiques pour améliorer l'efficacité du vaccin. Une composition à base de protéines donnée à titre d'exemple comprend un nanocorps de liaison à l'albumine et un domaine d'antigène peptidique. Un exemple de conjugué comprend la composition à base de protéines fixée à un médicament par l'intermédiaire d'un lieur. L'invention concerne également des procédés de fabrication et d'utilisation des compositions à base de protéines et des conjugués de celles-ci.
PCT/US2024/051131 2023-10-12 2024-10-11 Compositions de vaccin à nanocorps Pending WO2025081106A2 (fr)

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