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WO2025021277A1 - Agents et procédés d'administration ciblée de cytokines à des cellules immunitaires - Google Patents

Agents et procédés d'administration ciblée de cytokines à des cellules immunitaires Download PDF

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Publication number
WO2025021277A1
WO2025021277A1 PCT/EP2023/070312 EP2023070312W WO2025021277A1 WO 2025021277 A1 WO2025021277 A1 WO 2025021277A1 EP 2023070312 W EP2023070312 W EP 2023070312W WO 2025021277 A1 WO2025021277 A1 WO 2025021277A1
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WO
WIPO (PCT)
Prior art keywords
glu
arg
leu
moiety
particle
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/EP2023/070312
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English (en)
Inventor
Meike GANGLUFF
Ugur Sahin
Isabell Sofia KEIL
Mustafa DIKEN
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.)
TRON Translationale Onkologie an der Universitaetsmedizin der Johannes Gutenberg Universitaet Mainz gGmbH
Biontech SE
Original Assignee
TRON Translationale Onkologie an der Universitaetsmedizin der Johannes Gutenberg Universitaet Mainz gGmbH
Biontech SE
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Publication date
Application filed by TRON Translationale Onkologie an der Universitaetsmedizin der Johannes Gutenberg Universitaet Mainz gGmbH, Biontech SE filed Critical TRON Translationale Onkologie an der Universitaetsmedizin der Johannes Gutenberg Universitaet Mainz gGmbH
Priority to PCT/EP2023/070312 priority Critical patent/WO2025021277A1/fr
Priority to PCT/EP2024/070487 priority patent/WO2025021668A1/fr
Publication of WO2025021277A1 publication Critical patent/WO2025021277A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • A61K47/6849—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 receptor, a cell surface antigen or a cell surface determinant
    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons

Definitions

  • the invention relates to agents and methods for targeted delivery of RNA such as mRNA encoding a polypeptide comprising a cytokine or a functional variant thereof to immune cells for expression of the polypeptide.
  • Delivering RNA encoding a cytokine to immune cells may be useful for immunomodulation of immune cells, in particular for inducing proliferation of immune cells.
  • the invention involves a particle, and a targeting compound comprising a moiety incorporating into the particle, e.g., a hydrophobic moiety, and having a binding moiety covalently attached thereto.
  • the particle carries an RNA payload, i.e., RNA encoding a polypeptide comprising a cytokine or a functional variant thereof.
  • Targeting of an immune cell may be achieved by the direct or indirect binding of the targeting compound to cell surface antigens on the target immune cell of interest.
  • the binding moiety of the targeting compound binds to a cell surface antigen on a target immune cell, e.g., an immune effector cell, for targeting the particle carrying an RNA payload to target immune cells.
  • a target immune cell e.g., an immune effector cell
  • the binding moiety of the targeting compound may be constructs that have affinity for cell surface targets, e.g., membrane proteins, and include antibodies or antibody fragments.
  • the binding moiety of the targeting compound binds to a docking compound binding to a cell surface antigen on a target immune cell, e.g., an immune effector cell, for targeting the particle carrying an RNA payload to target immune cells.
  • the docking compound comprises a peptide or polypeptide.
  • the docking compound comprises a binding moiety binding to target immune cells (primary targeting moiety) and a further binding moiety binding to the binding moiety of the targeting compound.
  • the binding moiety of the targeting compound may bind to its binding moiety on the docking compound and then the primary targeting moiety may bind to a target antigen on target immune cells such as an antigen on immune effector cells to thereby precisely deliver an RNA payload to the target immune cells such as immune effector cells.
  • the present invention relates to an approach wherein particles comprising an RNA payload and a targeting compound are used.
  • the targeting compound comprises (i) a hydrophobic moiety for incorporation into the particles and (ii) a binding moiety covalently attached to the hydrophobic moiety for direct or indirect targeting of the particles to target immune cells and delivering the RNA payload to target immune cells.
  • Cell targeting may be achieved by the direct or indirect binding of the targeting compound to cell surface antigens on the target immune cell of interest.
  • particles comprising an RNA payload and a targeting compound are administered.
  • the binding moiety of the targeting compound binds to target immune cells, e.g., by binding to a cell surface antigen, thus resulting in cellular uptake of the RNA payload.
  • target immune cell binding moieties on the targeting compound are antibodies.
  • particles comprising an RNA payload and a targeting compound, and a docking compound that binds to target immune cells, e.g., by binding to a cell surface antigen are administered.
  • the targeting compound may be equipped with a binding moiety targeting a moiety on the docking compound.
  • a docking compound which is bound via a targeting compound to a particle comprising an RNA payload is administered.
  • the docking compound may bind to target immune cells, e.g., by binding to a cell surface antigen, thus resulting in cellular uptake of the RNA payload.
  • Common examples for pairs of interacting moieties on the targeting compound and on the docking compound are antibody/antigen systems.
  • Common examples for target immune cell binding moieties on the docking compound are antibodies.
  • the invention relates to agents and methods for targeted delivery of RNA encoding a polypeptide comprising a cytokine or a functional variant thereof, i.e., an RNA payload, to immune cells.
  • the agents and methods for targeted delivery of an RNA payload described herein may be used for generating in vitro/ex vivo or in vivo immune cells, e.g., immune effector cells, transfected with RNA encoding a polypeptide comprising a cytokine or a functional variant thereof.
  • immune cells transfected with RNA encoding a polypeptide comprising a cytokine or a functional variant thereof express the polypeptide.
  • Transfection is achieved using particles described herein comprising RNA encoding a polypeptide comprising a cytokine or a functional variant thereof and a targeting compound for targeting immune cells directly or via a docking compound binding to the targeting compound.
  • the particles may deliver the RNA to immune cells in vitro/ex vivo as well as in vivo.
  • Immune effector cells transfected as described herein are useful in the treatment of diseases wherein targeting cells such as diseased cells expressing an antigen such as a tumor antigen is beneficial.
  • the target cells may express the antigen on the cell surface for recognition by a CAR or in the context of MHC for recognition by a TCR.
  • Immune effector cells expressing an antigen receptor e.g., a CAR or TCR, targeting cells through binding to the antigen (or a procession product thereof) may be provided to a subject such as by administration of genetically modified immune effector cells to the subject or generation of genetically modified immune effector cells in the subject.
  • the immune effector cells are CD3+ T cells.
  • the target cell binding moiety (which may be present on the targeting compound or on the docking compound described herein) binds to the CD3 receptor on T cells.
  • the immune effector cells are CD8+ T cells. In some embodiments, the target cell binding moiety binds to the CD8 receptor on T cells. In some embodiments, the immune effector cells are CD4+ T cells. In some embodiments, the target cell binding moiety binds to the CD4 receptor on T cells.
  • the methods and agents described herein are, in particular, useful for the treatment of diseases characterized by diseased cells expressing an antigen the immune effector cells are directed to. In some embodiments, the immune effector cells by means of a CAR have a binding specificity for disease-associated antigen when present on diseased cells.
  • the immune effector cells by means of a TCR have a binding specificity for a procession product of disease-associated antigen when presented on diseased cells.
  • an immune effector cell is genetically modified to stably or transiently express an antigen receptor on its surface.
  • the invention relates to a particle comprising:
  • a moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag, wherein the particle carries RNA encoding a polypeptide comprising a cytokine or a functional variant thereof.
  • the moiety incorporating the targeting compound into the particle, and the moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag are linked by a linking moiety comprising a polymer P.
  • the targeting compound comprises the formula:
  • P is absent or comprises a polymer
  • L comprises a moiety incorporating the targeting compound into the particle
  • B comprises a moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag,
  • XI is absent or a first linking moiety
  • X2 is absent or a second linking moiety.
  • the particle comprises a lipid particle, a polymer particle, or a mixture thereof. In some embodiments, the particle comprises a lipid particle.
  • the moiety incorporating the targeting compound into the particle comprises a hydrophobic moiety
  • the moiety incorporating the targeting compound into the particle comprises a moiety selected from vitamin E, dialkylamine, diacylglyceride and ceramide.
  • the moiety incorporating the targeting compound into the particle comprises a C8-C24 hydrocarbon chain. In some embodiments, the moiety incorporating the targeting compound into the particle comprises two C8-C24 hydrocarbon chains.
  • the moiety incorporating the targeting compound into the particle comprises a lipid.
  • the moiety incorporating the targeting compound into the particle comprises a phospholipid.
  • the moiety incorporating the targeting compound into the particle comprises a moiety selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine), and mixtures thereof.
  • DSPE disearoylphosphatidylethanolamine
  • DPPE dipalmitoylphosphatidylethanolamine
  • DOPE dioleoylphosphatidylethanolamine
  • POPE palmitoyloleylphosphatidylethanolamine
  • P comprises a hydrophilic polymer.
  • P is selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), derivatives and combinations thereof.
  • PEG polyethylene glycol
  • pSar polysarcosine
  • POX polyoxazoline
  • POZ polyoxazine
  • pAEEA poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid
  • X2 comprises the reaction product of a thiol or cysteine reactive group with a thiol or cysteine group of a compound comprising the moiety B.
  • the thiol or cysteine reactive group comprises a maleimide group.
  • the targeting compound comprises the reaction product of 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)] with a compound comprising the formula SH(CH2)nC(O)-B, wherein n ranges from 1 to 5 and preferably n is 2.
  • the targeting compound comprises a compound of the formula:
  • the moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag comprises a moiety binding to a cell surface antigen on immune cells.
  • the moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag comprises a moiety selected from the group consisting of a tag and a moiety binding to a tag and the particle further comprises a docking compound comprising:
  • the docking compound comprises the formula:
  • B' comprises a moiety binding to the moiety selected from the group consisting of a tag and a moiety binding to a tag
  • X3 is absent or a linking moiety
  • B" comprises a moiety binding to a cell surface antigen on immune cells.
  • the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a tag and the moiety binding to the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a moiety binding to the tag.
  • the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a moiety binding to a tag and the moiety binding to the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a tag to which the moiety binding to a tag binds.
  • the docking compound comprises a peptide or polypeptide.
  • the moiety binding to a cell surface antigen on immune cells comprises a peptide or polypeptide. In some embodiments, the moiety binding to a cell surface antigen on immune cells comprises an antibody or antibody-like molecule.
  • the antibody-like molecule comprises an antibody fragment or DARPin.
  • the antibody-like molecule comprises a nanobody.
  • the immune cells comprise T cells.
  • the immune cells comprise CD8+ and/or CD4+ T cells.
  • the cell surface antigen on immune cells is characteristic for the immune cells.
  • the cell surface antigen on immune cells is selected from the group consisting of CD4, CD8, CD3, CD2, CD5, and CD127. In some embodiments, the cell surface antigen on immune cells is selected from the group consisting of CD4, CD8 and CD3 In some embodiments, the moiety binding to a tag comprises a peptide or polypeptide.
  • the moiety binding to a tag comprises an antibody or antibody-like molecule.
  • the antibody-like molecule comprises an antibody fragment or DARPin.
  • the antibody-like molecule comprises a nanobody.
  • the tag comprises a peptide or polypeptide.
  • the tag comprises a peptide tag.
  • the tag comprises an ALFA-tag.
  • the tag comprises an ALFA-tag and the moiety binding to the tag comprises a VHH domain comprising the CDR1 sequence VTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY.
  • the particle is a non-viral particle.
  • the particle is a nanoparticle.
  • the particle is a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • the cytokine comprises an interleukin.
  • the cytokine comprises interleukin 2.
  • the invention relates to a composition, e.g., a pharmaceutical composition, comprising particles as described herein.
  • the invention relates to a method for delivering a polypeptide comprising a cytokine or a functional variant thereof to immune cells expressing a cell surface antigen, comprising adding to the immune cells a composition comprising particles, wherein a particle comprises:
  • a moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag, wherein the particle carries RNA encoding a polypeptide comprising a cytokine or a functional variant thereof.
  • the moiety incorporating the targeting compound into the particle, and the moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag are linked by a linking moiety comprising a polymer P.
  • the targeting compound comprises the formula:
  • P is absent or comprises a polymer
  • L comprises a moiety incorporating the targeting compound into the particle
  • B comprises a moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag,
  • XI is absent or a first linking moiety
  • X2 is absent or a second linking moiety.
  • the particle comprises a lipid particle, a polymer particle, or a mixture thereof.
  • the particle comprises a lipid particle.
  • the moiety incorporating the targeting compound into the particle comprises a hydrophobic moiety
  • the moiety incorporating the targeting compound into the particle comprises a moiety selected from vitamin E, dialkylamine, diacylglyceride and ceramide.
  • the moiety incorporating the targeting compound into the particle comprises a C8-C24 hydrocarbon chain. In some embodiments, the moiety incorporating the targeting compound into the particle comprises two C8-C24 hydrocarbon chains.
  • the moiety incorporating the targeting compound into the particle comprises a lipid.
  • the moiety incorporating the targeting compound into the particle comprises a phospholipid.
  • the moiety incorporating the targeting compound into the particle comprises a moiety selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine), and mixtures thereof.
  • DSPE disearoylphosphatidylethanolamine
  • DPPE dipalmitoylphosphatidylethanolamine
  • DOPE dioleoylphosphatidylethanolamine
  • POPE palmitoyloleylphosphatidylethanolamine
  • P comprises a hydrophilic polymer.
  • P is selected from the group consisting of poly(ethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), derivatives and combinations thereof.
  • PEG poly(ethylene glycol)
  • pSar polysarcosine
  • POX polyoxazoline
  • POZ polyoxazine
  • pAEEA poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid
  • X2 comprises the reaction product of a thiol or cysteine reactive group with a thiol or cysteine group of a compound comprising the moiety B.
  • the thiol or cysteine reactive group comprises a maleimide group.
  • the targeting compound comprises the reaction product of 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)] with a compound comprising the formula SH(CH2)nC(O)-B, wherein n ranges from 1 to 5 and preferably n is 2.
  • the targeting compound comprises a compound of the formula:
  • the moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag comprises a moiety binding to a cell surface antigen on immune cells.
  • the moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag comprises a moiety selected from the group consisting of a tag and a moiety binding to a tag and the particle further comprises a docking compound comprising:
  • the docking compound comprises the formula:
  • B' comprises a moiety binding to the moiety selected from the group consisting of a tag and a moiety binding to a tag
  • X3 is absent or a linking moiety
  • B" comprises a moiety binding to a cell surface antigen on immune cells.
  • the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a tag and the moiety binding to the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a moiety binding to the tag.
  • the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a moiety binding to a tag and the moiety binding to the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a tag to which the moiety binding to a tag binds.
  • the docking compound comprises a peptide or polypeptide.
  • the moiety bindingto a cell surface antigen on immune cells comprises a peptide or polypeptide. In some embodiments, the moiety binding to a cell surface antigen on immune cells comprises an antibody or antibody-like molecule.
  • the antibody-like molecule comprises an antibody fragment or DARPin.
  • the antibody-like molecule comprises a nanobody
  • the moiety binding to a cell surface antigen on immune cells binds to the cell surface antigen expressed by the immune cells.
  • the immune cells comprise T cells.
  • the immune cells comprise CD8+ and/or CD4+ T cells.
  • the cell surface antigen on immune cells is characteristic for the immune cells.
  • the cell surface antigen on immune cells is selected from the group consisting of CD4, CD8, CD3, CD2, CD5, and CD127. In some embodiments, the cell surface antigen on immune cells is selected from the group consisting of CD4, CD8 and CD3 In some embodiments, the moiety binding to a tag comprises a peptide or polypeptide.
  • the moiety binding to a tag comprises an antibody or antibody-like molecule.
  • the antibody-like molecule comprises an antibody fragment or DARPin.
  • the antibody-like molecule comprises a nanobody.
  • the tag comprises a peptide or polypeptide.
  • the tag comprises a peptide tag.
  • the tag comprises an ALFA-tag.
  • the tag comprises an ALFA-tag and the moiety binding to the tag comprises a VHH domain comprising the CDR1 sequence VTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY.
  • the particle is a non-viral particle.
  • the particle is a nanoparticle.
  • the particle is a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • the cytokine comprises an interleukin. In some embodiments, the cytokine comprises interleukin 2.
  • the method is a method for immunomodulation of immune cells.
  • the method is a method for inducing proliferation of immune cells.
  • the immune cells are present ex vivo or in vitro.
  • the immune cells are present in a subject and the method comprises administering the composition to the subject.
  • the invention relates to a method for inducing proliferation of immune cells, comprising adding to the immune cells a composition comprising particles, wherein a particle comprises:
  • a moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag, wherein the particle carries RNA encoding a polypeptide comprising a cytokine or a functional variant thereof.
  • the moiety incorporating the targeting compound into the particle, and the moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag are linked by a linking moiety comprising a polymer P.
  • the targeting compound comprises the formula:
  • P is absent or comprises a polymer
  • L comprises a moiety incorporating the targeting compound into the particle
  • B comprises a moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag,
  • XI is absent or a first linking moiety
  • the particle comprises a lipid particle, a polymer particle, or a mixture thereof.
  • the particle comprises a lipid particle.
  • the moiety incorporating the targeting compound into the particle comprises a hydrophobic moiety
  • the moiety incorporating the targeting compound into the particle comprises a moiety selected from vitamin E, dialkylamine, diacylglyceride and ceramide.
  • the moiety incorporating the targeting compound into the particle comprises a C8-C24 hydrocarbon chain. In some embodiments, the moiety incorporating the targeting compound into the particle comprises two C8-C24 hydrocarbon chains.
  • the moiety incorporating the targeting compound into the particle comprises a lipid.
  • the moiety incorporating the targeting compound into the particle comprises a phospholipid.
  • the moiety incorporating the targeting compound into the particle comprises a moiety selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine), and mixtures thereof.
  • DSPE disearoylphosphatidylethanolamine
  • DPPE dipalmitoylphosphatidylethanolamine
  • DOPE dioleoylphosphatidylethanolamine
  • POPE palmitoyloleylphosphatidylethanolamine
  • P comprises a hydrophilic polymer.
  • P is selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), derivatives and combinations thereof.
  • PEG polyethylene glycol
  • pSar polysarcosine
  • POX polyoxazoline
  • POZ polyoxazine
  • pAEEA poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid
  • X2 comprises the reaction product of a thiol or cysteine reactive group with a thiol or cysteine group of a compound comprising the moiety B.
  • the thiol or cysteine reactive group comprises a maleimide group.
  • the targeting compound comprises the reaction product of 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)] with a compound comprising the formula SH(CH2)nC(O)-B, wherein n ranges from 1 to 5 and preferably n is 2.
  • the targeting compound comprises a compound of the formula:
  • the moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag comprises a moiety binding to a cell surface antigen on immune cells.
  • the moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag comprises a moiety selected from the group consisting of a tag and a moiety binding to a tag and the particle further comprises a docking compound comprising:
  • the docking compound comprises the formula:
  • B' comprises a moiety binding to the moiety selected from the group consisting of a tag and a moiety binding to a tag
  • X3 is absent or a linking moiety
  • B" comprises a moiety binding to a cell surface antigen on immune cells.
  • the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a tag and the moiety binding to the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a moiety binding to the tag.
  • the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a moiety binding to a tag and the moiety binding to the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a tag to which the moiety binding to a tag binds.
  • the docking compound comprises a peptide or polypeptide.
  • the moiety bindingto a cell surface antigen on immune cells comprises a peptide or polypeptide.
  • the moiety bindingto a cell surface antigen on immune cells comprises an antibody or antibody-like molecule.
  • the antibody-like molecule comprises an antibody fragment or DARPin.
  • the antibody-like molecule comprises a nanobody.
  • the moiety binding to a cell surface antigen on immune cells binds to a cell surface antigen expressed by the immune cells.
  • the immune cells comprise T cells.
  • the immune cells comprise CD8+ and/or CD4+ T cells.
  • the cell surface antigen on immune cells is characteristic for the immune cells.
  • the cell surface antigen on immune cells is selected from the group consisting of CD4, CD8, CD3, CD2, CD5, and CD127. In some embodiments, the cell surface antigen on immune cells is selected from the group consisting of CD4, CD8 and CD3 In some embodiments, the moiety binding to a tag comprises a peptide or polypeptide.
  • the moiety binding to a tag comprises an antibody or antibody-like molecule.
  • the antibody-like molecule comprises an antibody fragment or DARPin.
  • the antibody-like molecule comprises a nanobody.
  • the tag comprises a peptide or polypeptide.
  • the tag comprises a peptide tag.
  • the tag comprises an ALFA-tag.
  • the tag comprises an ALFA-tag and the moiety binding to the tag comprises a VHH domain comprising the CDR1 sequence VTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY.
  • the particle is a non-viral particle.
  • the particle is a nanoparticle. In some embodiments, the particle is a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • the cytokine comprises an interleukin.
  • the cytokine comprises interleukin 2.
  • the immune cells are present ex vivo or in vitro.
  • the immune cells are present in a subject and the method comprises administering the composition to the subject.
  • the invention relates to a method for treating a subject comprising:
  • the invention relates to a method for treating a subject comprising administering to the subject a composition comprising particles described herein.
  • the invention relates to an agent or composition described herein for use in a method for treating a subject described herein.
  • Figure 1 Physicochemical characterization of DODMA-based lipid nanoparticles (LNPs) containing Thyl.l/hlL-2 mRNA mix.
  • LNPs DODMA-based lipid nanoparticles
  • RNA mix 1:1 w/w, with or without ahCD3 VHH X NbALFA ligand.
  • FIG. 2 In vitro transfection efficiency of lipid nanoparticles (LNPs) containing Thyl.l/hlL-2 mRNA mix.
  • Human PBMCs were transfected with respective LNPs (2000 ng RNA total; 250 ng Thyl.l RNA (A), 1750 ng hlL-2 RNA (B); RNA mix 1:8 w/w; LNPs were formulated with or without ahCD3 VHH X NbALFA ligand).
  • PMA/lono was supplemented to medium as positive control, lx HBT buffer (10 mM HEPES, 10 w/v % Trehalose, pH 7.1) served as negative control.
  • FIG. 3 Physicochemical characterization of HY501-based lipid nanoparticles (LNPs) containing Thyl.l/Luc and Thyl.l/hlL-2 mRNA mix. Respective LNPs were formulated with an RNA mix of 1:1 w/w with or without ahCD3 VHH X NbALFA ligand. Size (A) and PDI (B) measured via dynamic light scattering. RNA integrity performed by Fragment Analyzer (C). Zeta Potential measured via Zetasizer (D). All physicochemical parameters in the expected range. Successful RNA incorporation was verified via agarose gel electrophoresis (not shown).
  • FIG. 4 Ex vivo detection of hlL-2 in supernatant of splenocytes from transgenic B6-hCD3EDG mice by ahCD3 VHH X NbALFA-LNPs containing hlL-2 encoded mRNA.
  • Splenocytes of transgenic B6-hCD3EDG mice were transfected with respective LNPs (2000 ng RNA total, LNPs were formulated with an RNA mix of 1:1 w/w with or without ahCD3 VHH X NbALFA ligand).
  • hlL-2 mRNA containing LNPs lead to hlL-2 serum detection at 48 h.
  • Calculated serum hlL-2 concentration analyzed by singleplex assay (hlL-2 MSD). Two representative mice are shown.
  • FIG. 6 T cell-restricted activation and proliferation by ahCD3 VHH X NbALFA-LNPs containing hlL-2 encoded mRNA in blood at 48 h.
  • mice Data are presented as mean ⁇ S.D., analyzed by a two-way ANOVA with Sidak's multiple comparison test, *P ⁇ 0.1, **P ⁇ 0.01, ***P ⁇ 0.001, ****p ⁇ 0.0001. Three representative mice are shown.
  • FIG. 7 Lymphocyte cell count sustained high in blood at 96 h after treatment with hlL-2 mRNA containing ahCD3 VHH X NbALFA-LNP.
  • Count of lymphocyte sub-populations analyzed by flow cytometry. Data are presented as mean ⁇ S.D., analyzed by a two-way ANOVA with Sidak's multiple comparison test, *P ⁇ 0.1, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001.
  • the term "about” denotes an interval of accuracy that the person of ordinary skill will understand to still ensure the technical effect of the feature in question.
  • the term typically indicates deviation from the indicated numerical value by +10%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, ⁇ 1%, ⁇ 0.9%, ⁇ 0.8%, ⁇ 0.7%, ⁇ 0.6%, ⁇ 0.5%, ⁇ 0.4%, ⁇ 0.3%, ⁇ 0.2%, ⁇ 0.1%, ⁇ 0.05%, and for example ⁇ 0.01%.
  • "about” indicates deviation from the indicated numerical value by ⁇ 10%.
  • "about” indicates deviation from the indicated numerical value by ⁇ 5%.
  • “about” indicates deviation from the indicated numerical value by ⁇ 4%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 3%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 2%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 1%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.9%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.8%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.7%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.6%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.5%.
  • “about” indicates deviation from the indicated numerical value by ⁇ 0.4%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.3%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.2%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.1%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.05%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.01%. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect.
  • Physiological pH refers to a pH of about 7.4. In some embodiments, physiological pH is from 7.3 to 7.5. In some embodiments, physiological pH is from 7.35 to 7.45. In some embodiments, physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.
  • % w/v refers to weight by volume percent, which is a unit of concentration measuring the amount of solute in grams (g) expressed as a percent of the total volume of solution in milliliters (mL).
  • % by weight refers to weight percent, which is a unit of concentration measuring the amount of a substance in grams (g) expressed as a percent of the total weight of the total composition in grams (g).
  • mol % is defined as the ratio of the number of moles of one component to the total number of moles of all components, multiplied by 100.
  • mol % of the total lipid is defined as the ratio of the number of moles of one lipid component to the total number of moles of all lipids, multiplied by 100.
  • total lipid includes lipids and lipid- like material.
  • ionic strength refers to the mathematical relationship between the number of different kinds of ionic species in a particular solution and their respective charges. Thus, ionic strength I is represented mathematically by the formula: in which c is the molar concentration of a particular ionic species and z the absolute value of its charge. The sum 1 is taken over all the different kinds of ions (i) in solution.
  • the term "ionic strength" in some embodiments relates to the presence of monovalent ions.
  • divalent ions in particular divalent cations
  • their concentration or effective concentration (presence of free ions) due to the presence of chelating agents is, in some embodiments, sufficiently low so as to prevent degradation of a nucleic acid.
  • the concentration or effective concentration of divalent ions is below the catalytic level for hydrolysis of the phosphodiester bonds between nucleotides such as RNA nucleotides.
  • the concentration of free divalent ions is 20 pM or less. In some embodiments, there are no or essentially no free divalent ions.
  • Oleality refers to the concentration of a particular solute expressed as the number of osmoles of solute per kilogram of solvent.
  • lyophilizing refers to the freeze-drying of a substance by freezing it and then reducing the surrounding pressure (e.g., below 15 Pa, such as below 10 Pa, below 5 Pa, or 1 Pa or less) to allow the frozen medium in the substance to sublimate directly from the solid phase to the gas phase.
  • surrounding pressure e.g., below 15 Pa, such as below 10 Pa, below 5 Pa, or 1 Pa or less
  • spray-drying refers to spray-drying a substance by mixing (heated) gas with a fluid that is atomized (sprayed) within a vessel (spray dryer), where the solvent from the formed droplets evaporates, leading to a dry powder.
  • the term "reconstitute” relates to adding a solvent such as water to a dried product to return it to a liquid state such as its original liquid state.
  • recombinant in the context of the present disclosure means “made through genetic engineering".
  • a "recombinant object” in the context of the present disclosure is not occurring naturally.
  • naturally occurring refers to the fact that an object can be found in nature.
  • a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.
  • found in nature means "present in nature” and includes known objects as well as objects that have not yet been discovered and/or isolated from nature, but that may be discovered and/or isolated in the future from a natural source.
  • room temperature and “ambient temperature” are used interchangeably herein and refer to temperatures from at least about 15°C, e.g., from about 15°C to about 35°C, from about 15°C to about 30°C, from about 15°C to about 25°C, or from about 17°C to about 22°C. Such temperatures will include 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C and 22°C.
  • EDTA refers to ethylenediaminetetraacetic acid disodium salt. All concentrations are given with respect to the EDTA disodium salt.
  • cryoprotectant relates to a substance that is added to a formulation in order to protect the active ingredients during the freezing stages.
  • lyoprotectant relates to a substance that is added to a formulation in order to protect the active ingredients during the drying stages.
  • peptide refers to substances which comprise about two or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100 or about 150, consecutive amino acids linked to one another via peptide bonds.
  • polypeptide refers to large peptides, in particular peptides having at least about 151 amino acids.
  • eptides and “polypeptides” are both protein molecules. Thus, the terms “peptide”, “protein” and “polypeptide” are used herein usually as synonyms.
  • Peptides and polypeptides disclosed herein may comprise a linear or a cyclized peptide sequence.
  • the peptides disclosed herein comprises at least one cyclic portion, i.e., a polypeptide chain that contains a circular sequence of bonds that is referred to herein as a "cyclic peptide.”
  • the circular sequence can occur through a connection between the amino and carboxyl ends of the peptide; a connection between the amino end and a side chain; a connection between the carboxyl end and a side chain; or a connection between two side chains including sulfur groups of two cysteine amino acids by forming a disulfide bond, or more complicated arrangements.
  • the peptides and polypeptides disclosed herein are composed of naturally occurring amino acids, non-naturally occurring amino acids, amino acid derivatives and non-amino acid components, or a mixture thereof.
  • the peptides and polypeptides disclosed herein comprise amino acid mimetics and amino acid analogs.
  • the peptides and polypeptides disclosed herein comprise non-naturally occurring amino acid sequences that are resistant to enzymatic cleavage.
  • one or more positions of a peptide or polypeptide disclosed herein are substituted with a non-naturally occurring amino acid.
  • the substituted amino acid is chemically related to the original residue (e.g., aliphatic, charged, basic, acidic, aromatic, hydrophilic) or an isostere of the original residue.
  • amino acid refers to a compound and/or substance that can be, is, or has been incorporated into a peptide, e.g., through formation of one or more peptide bonds.
  • an amino acid has the general structure H2N-C(H)(R)-COOH.
  • an amino acid is a naturally-occurring amino acid.
  • an amino acid is a non-natural amino acid.
  • an amino acid is a D-amino acid.
  • an amino acid is an L-amino acid.
  • Standard amino acid refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides and polypeptides.
  • Nonstandard amino acid refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source.
  • an amino acid, including a carboxy- and/or amino-terminal amino acid in a peptide or polypeptide, can contain a structural modification as compared with the general structure above.
  • an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and/or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and/or the hydroxyl group) as compared with the general structure.
  • such modification may, for example, alter the circulating half-life of a peptide or polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid.
  • such modification does not significantly alter a relevant activity of a peptide or polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid.
  • the term "amino acid" may be used to refer to a free amino acid. In some embodiments it may be used to refer to an amino acid residue of a peptide or polypeptide.
  • amino acids are L-amino acids while D-amino acids are denoted by the prefix "D”.
  • D D-amino acids
  • the prefix "homo” or “h” designates an a-amino acid that is otherwise similar to one of the common ones, but that contains one more methylene group in the carbon chain.
  • Orn means ornithine or 2,5-diaminopentanoic acid
  • Dab means 2,4- diaminobutanoic acid
  • Dap means 2,3-diaminopropanoic acid
  • hLys means 2,7- diaminoheptanoic acid
  • hCys means 2-amino-4-mercaptobutanoic acid
  • Pen means penicillamine or 2-amino-3-methyl-3-sulfanylbutanoic acid.
  • non-peptide linkages and other chemical modification may also be possible to include non-peptide linkages and other chemical modification.
  • part or all of the peptide or polypeptide may be synthesized as a peptidomimetic, e.g., a peptoid (see, e.g., Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89:9367-71 and Horwell (1995) Trends Biotechnol.13:132-4).
  • a peptide or polypeptide may include one or more (e.g., all) non-hydrolyzable bonds. Many non-hydrolyzable peptide bonds are known in the art, along with procedures for synthesis of peptides containing such bonds.
  • non- hydrolyzable bonds include -[CH2NH]- reduced amide peptide bonds, -[COCH2]- ketomethylene peptide bonds, -(CH(CN)NH]- (cyanomethylene)amino peptide bonds, - (CH2CH(OH)]- hydroxyethylene peptide bonds, -[CH2O]- oxymethylene peptide bonds, and - [CH2S]- thiomethylene peptide bonds (see e.g., U.S. Pat. No. 6,172,043).
  • amide as used herein, represents a group of formula "-NHC(O)-”.
  • thioamide represents a group of formula "-NHC(S)-”.
  • disulfide bond includes the covalent bond formed between two sulfur atoms.
  • the amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group.
  • ether refers to a group or compound having an oxygen between two carbon atoms.
  • thioether refers to a group or compound having a sulfur between two carbon atoms.
  • thioester refers to the group -C(O)S-.
  • triazole refers to chemical compounds that incorporate in their structure any heterocyclic structure having a five-membered ring of two carbon atoms and three nitrogen atoms (e.g., 1,2,3-triazole).
  • portion refers to a fraction. With respect to a particular structure such as an amino acid sequence or protein the term “portion” thereof may designate a continuous or a discontinuous fraction of said structure.
  • part and fragment are used interchangeably herein and refer to a continuous element.
  • a part of a structure such as an amino acid sequence or protein refers to a continuous element of said structure.
  • the term “part” means a portion of the composition.
  • a part of a composition may be any portion from 0.1% to 99.9% (such as 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%) of said composition.
  • “Fragment” with reference to an amino acid sequence (peptide or polypeptide), relates to a part of an amino acid sequence, i.e., a sequence which represents the amino acid sequence shortened at the N-terminus and/or C-terminus.
  • a fragment shortened at the C-terminus is obtainable, e.g., by translation of a truncated open reading frame that lacks the 3'-end of the open reading frame.
  • a fragment shortened at the N-terminus is obtainable, e.g., by translation of a truncated open reading frame that lacks the 5'-end of the open reading frame, as long as the truncated open reading frame comprises a start codon that serves to initiate translation.
  • a fragment of an amino acid sequence comprises, e.g., at least 50 %, at least 60 %, at least 70 %, at least 80%, at least 90% of the amino acid residues from an amino acid sequence.
  • a fragment of an amino acid sequence comprises, e.g., at least 6, in particular at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from an amino acid sequence.
  • a fragment of an amino acid sequence comprises, e.g., a sequence of up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55, consecutive amino acids of the amino acid sequence.
  • Variant as used herein and with reference to an amino acid sequence (peptide or polypeptide), is meant an amino acid sequence that differs from a parent amino acid sequence by virtue of at least one amino acid (e.g., a different amino acid, or a modification of the same amino acid).
  • the parent amino acid sequence may be a naturally occurring or wild type (WT) amino acid sequence, or may be a modified version of a wild type amino acid sequence.
  • the variant amino acid sequence has at least one amino acid difference as compared to the parent amino acid sequence, e.g., from 1 to about 20 amino acid differences, such as from 1 to about 10 or from 1 to about 5 amino acid differences compared to the parent.
  • wild type or “WT” or “native” as used herein and with reference to an amino acid sequence is meant an amino acid sequence that is found in nature, including allelic variations.
  • a wild type amino acid sequence, peptide or polypeptide has an amino acid sequence that has not been intentionally modified.
  • variants of an amino acid sequence may comprise amino acid insertion variants, amino acid addition variants, amino acid deletion variants and/or amino acid substitution variants.
  • variant includes all mutants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, in particular those which are naturally occurring.
  • variant includes, in particular, fragments of an amino acid sequence.
  • Amino acid insertion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence.
  • amino acid sequence variants having an insertion one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible.
  • Amino acid addition variants comprise amino- and/or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids.
  • Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions may be in any position of the protein.
  • Amino acid deletion variants that comprise the deletion at the N-terminal and/or C-terminal end of the protein are also called N-terminal and/or C- terminal truncation variants.
  • Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Preference is given to the modifications being in positions in the amino acid sequence which are not conserved between homologous peptides or polypeptides and/or to replacing amino acids with other ones having similar properties.
  • amino acid changes in peptide and polypeptide variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids.
  • a conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.
  • Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.
  • conservative amino acid substitutions include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • the degree of similarity such as identity between a given amino acid sequence and an amino acid sequence which is a variant of said given amino acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • the degree of similarity or identity is given for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence.
  • the degree of similarity or identity is given, e.g., for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments continuous amino acids.
  • the degree of similarity or identity is given for the entire length of the reference amino acid sequence.
  • the alignment for determining sequence similarity, such as sequence identity can be done with art known tools, such as using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.
  • Sequence similarity indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions.
  • Sequence identity between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences.
  • Sequnce identity between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.
  • % identical and % identity are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or "window of comparison", in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of algorithms, e.g., the local homology algorithm by Smith and Waterman, 1981, Ads App. Math.
  • NCBI National Center for Biotechnology Information
  • the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match/Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used.
  • the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment.
  • Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.
  • the degree of similarity or identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence.
  • the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments continuous nucleotides.
  • the degree of similarity or identity is given for the entire length of the reference sequence.
  • Homologous amino acid sequences exhibit according to the disclosure at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and, e.g., at least 95%, at least 98 or at least 99% identity of the amino acid residues.
  • amino acid sequence variants described herein may readily be prepared by the skilled person, for example, by recombinant DNA manipulation.
  • the manipulation of DNA sequences for preparing peptides or polypeptides having substitutions, additions, insertions or deletions, is described in detail in Molecular Cloning: A Laboratory Manual, 4th Edition, M.R. Green and J. Sambrook et al. (1989), eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012, for example.
  • the peptides, polypeptides and amino acid variants described herein may be readily prepared with the aid of known peptide synthesis techniques such as, for example, by solid phase synthesis and similar methods.
  • a fragment or variant of an amino acid sequence is a "functional fragment” or “functional variant”.
  • the term "functional fragment” or “functional variant” of an amino acid sequence relates to any fragment or variant exhibiting one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., it is functionally equivalent.
  • sequences of binding agents such as antibodies, one particular function is one or more binding activities displayed by the amino acid sequence from which the fragment or variant is derived.
  • the modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence.
  • the function of the functional fragment or functional variant may be reduced but still significantly present, e.g., function of the functional fragment or functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the parent molecule or sequence. However, in other embodiments, function of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.
  • amino acid sequence (peptide or polypeptide) "derived from” a designated amino acid sequence (peptide or polypeptide) refers to the origin of the first amino acid sequence.
  • the amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof.
  • Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof.
  • sequences suitable for use herein may be altered such that they vary in sequence from the naturally occurring or native sequences from which they were derived, while retaining the desirable activity of the native sequences.
  • isolated means removed (e.g., purified) from the natural state or from an artificial composition, such as a composition from a production process.
  • a nucleic acid, peptide or polypeptide naturally present in a living animal is not “isolated”, but the same nucleic acid, peptide or polypeptide partially or completely separated from the coexisting materials of its natural state is “isolated”.
  • An isolated nucleic acid, peptide or polypeptide can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • bind or “binding” relates to the non-covalent interaction with a target.
  • the term “bind” or “binding” relates to a specific binding.
  • specific binding or “specifically binds”, as used herein, is meant a molecule such as an antibody or antigen receptor which recognizes a specific target molecule, but does not substantially recognize or bind other molecules in a sample or in a subject.
  • an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific.
  • an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific.
  • the terms “specific binding” or “specifically binds”, can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope "A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled "A” and the antibody, will reduce the amount of labeled A bound to the antibody.
  • a particular structure e.g., an antigenic determinant or epitope
  • binding typically is a binding with an affinity corresponding to a KD of about 10' 7 M or less, such as about 10' 8 M or less, such as about 10' 9 M or less, about 10 10 M or less, or about 10 11 M or even less, when determined using Bio-Layer Interferometry (BLI), or, for instance, when determined using surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument.
  • BLI Bio-Layer Interferometry
  • SPR surface plasmon resonance
  • a binding moiety or agent binds to a predetermined target with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100-fold lower, for instance at least 1,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its affinity for binding to a non-specific target (e.g., BSA, casein).
  • ka ka
  • sec 1 refers to the dissociation rate constant of a particular interaction, e.g., antibody-antigen interaction. Said value is also referred to as the k O ff value.
  • KD (M), as used herein, refers to the dissociation equilibrium constant of a particular interaction, e.g., antibody-antigen interaction.
  • binding or “binding” and “target” or “targeting” are used interchangeably herein.
  • the term “genetic modification” or simply “modification” includes the transfection of cells with nucleic acid.
  • the term “transfection” relates to the introduction of nucleic acids, e.g., DNA and/or RNA, into a cell.
  • the term “transfection” also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such cell, wherein the cell may be present in a subject, e.g., a patient, or the cell may be in vitro, e.g., outside of a patient.
  • a cell for transfection of a nucleic acid described herein can be present in vitro or in vivo, e.g.
  • transfection can be transient or stable.
  • the transfected genetic material is only transiently expressed.
  • RNA can be transfected into cells to transiently express its coded protein. Since the nucleic acid introduced in the transfection process is usually not integrated into the nuclear genome, the foreign nucleic acid will be diluted through mitosis or degraded. Cells allowing episomal amplification of nucleic acids greatly reduce the rate of dilution. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, a stable transfection must occur.
  • Such stable transfection can be achieved by using virus-based systems or transposon-based systems for transfection, for example.
  • cells that are genetically modified to express an antigen receptor are stably transfected with nucleic acid encoding the antigen receptor.
  • RNA can be transfected into cells to transiently express its coded protein.
  • fusion protein refers to a polypeptide or protein comprising two or more subunits.
  • the fusion protein is a translational fusion between the two or more subunits.
  • the translational fusion may be generated by genetically engineering the coding nucleotide sequence for one subunit in a reading frame with the coding nucleotide sequence of a further subunit. Subunits may be interspersed by a linker.
  • endogenous refers to any material from or produced inside an organism, cell, tissue or system.
  • exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
  • autologous transplant refers to a transplant of tissue or organs derived from the same subject. Such procedures are advantageous because they overcome the immunological barrier which otherwise results in rejection.
  • allogeneic is used to describe anything that is derived from different individuals of the same species. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical.
  • genotypeic is used to describe anything that is derived from individuals or tissues having identical genotypes, i.e., identical twins or animals of the same inbred strain, or their tissues.
  • heterologous is used to describe something consisting of multiple different elements. As an example, the transfer of one individual's bone marrow into a different individual constitutes a heterologous transplant.
  • a heterologous gene is a gene derived from a source other than the subject.
  • a nucleic acid encoding a peptide or polypeptide is taken up by or introduced, i.e. transfected or transduced, into a cell which cell may be present in vitro or in a subject, resulting in expression of said peptide or polypeptide.
  • the cell may, e.g., express the encoded peptide or polypeptide intracellularly (e.g. in the cytoplasm and/or in the nucleus), may secrete the encoded peptide or polypeptide, and/or may express it on the surface.
  • nucleic acid expressing and “nucleic acid encoding” or similar terms are used interchangeably herein and with respect to a particular peptide or polypeptide mean that the nucleic acid, if present in the appropriate environment, e.g. within a cell, can be expressed to produce said peptide or polypeptide.
  • expression includes the transcription and/or translation of a particular nucleotide sequence.
  • transcription relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA (especially mRNA). Subsequently, the RNA may be translated into peptide or polypeptide.
  • RNA With respect to RNA, the term "expression” or “translation” relates to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino acids to make a peptide or polypeptide.
  • a medical preparation, in particular kit, comprising, for example, particles described herein may comprise instructional material or instructions.
  • "instructional material” or “instructions” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the present disclosure.
  • the instructional material of the kit of the present disclosure may, for example, be affixed to a container which contains the compositions/formulations of the present disclosure or be shipped together with a container which contains the compositions/formulations. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compositions be used cooperatively by the recipient.
  • average diameter refers to the mean hydrodynamic diameter of particles as measured by dynamic light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Z ave rage with the dimension of a length, and the polydispersity index (PDI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321).
  • PDI polydispersity index
  • the "polydispersity index” is calculated based on dynamic light scattering measurements by the so-called cumulant analysis as mentioned in the definition of the "average diameter". Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of nanoparticles.
  • the "radius of gyration" (abbreviated herein as R g ) of a particle about an axis of rotation is the radial distance of a point from the axis of rotation at which, if the whole mass of the particle is assumed to be concentrated, its moment of inertia about the given axis would be the same as with its actual distribution of mass.
  • R g is the root mean square distance of the particle's components from either its center of mass or a given axis.
  • R g is the square-root of the mass average of Si 2 over all mass elements and can be calculated as follows:
  • the radius of gyration can be determined or calculated experimentally, e.g., by using light scattering.
  • the structure function S is defined as follows: wherein N is the number of components (Guinier's law).
  • the "hydrodynamic radius” (which is sometimes called “Stokes radius” or “Stokes-Einstein radius”) of a particle is the radius of a hypothetical hard sphere that diffuses at the same rate as said particle.
  • the hydrodynamic radius is related to the mobility of the particle, taking into account not only size but also solvent effects. For example, a smaller charged particle with stronger hydration may have a greater hydrodynamic radius than a larger charged particle with weaker hydration. This is because the smaller particle drags a greater number of water molecules with it as it moves through the solution.
  • the hydrodynamic radius may be defined by the Stokes-Einstein equation: wherein ke, is the Boltzmann constant; 7 is the temperature; q is the viscosity of the solvent; and D is the diffusion coefficient.
  • the diffusion coefficient can be determined experimentally, e.g., by using dynamic light scattering (DLS).
  • one procedure to determine the hydrodynamic radius of a particle or a population of particles is to measure the DLS signal of said particle or population of particles (such as DLS signal of particles contained in a sample or control composition as disclosed herein or the DLS signal of a particle peak obtained from subjecting such a sample or control composition to field-flow fractionation).
  • light scattering refers to the physical process where light is forced to deviate from a straight trajectory by one or more paths due to localized nonuniformities in the medium through which the light passes.
  • UV means ultraviolet and designates a band of the electromagnetic spectrum with a wavelength from 10 nm to 400 nm, i.e., shorter than that of visible light but longer than X- rays.
  • multi-angle light scattering or “MALS” as used herein relates to a technique for measuring the light scattered by a sample into a plurality of angles.
  • Multi-angle means in this respect that scattered light can be detected at different discrete angles as measured, for example, by a single detector moved over a range including the specific angles selected or an array of detectors fixed at specific angular locations.
  • the light source used in MALS is a laser source (MALLS: multi-angle laser light scattering).
  • the Zimm plot is a graphical presentation using the following equation: wherein c is the mass concentration of the particles in the solvent (g/mL); A2 is the second virial coefficient (mol-mL/g 2 ); P(&) is a form factor relating to the dependence of scattered light intensity on angle; ft?
  • K* is an optical constant that is equal to 4n 2 q 0 (dn/dc) 2 Ao’ 4 A/A S
  • q 0 is the refractive index of the solvent at the incident radiation (vacuum) wavelength
  • Ao is the incident radiation (vacuum) wavelength (nm)
  • A/A is Avogadro's number (mol x )
  • dn/dc is the differential refractive index increment (mL/g) (cf., e.g., Buchholz et al. (Electrophoresis 22 (2001), 4118-4128); B.H. Zimm (J. Chem. Phys. 13 (1945), 141; P.
  • the Berry plot is calculated using the following term or the reciprocal thereof: wherein c, Ra and K* are as defined above.
  • the Debye plot is calculated using the following term or the reciprocal thereof: wherein c, R$ and K* are as defined above.
  • DLS dynamic light scattering
  • a monochromatic light source usually a laser
  • the scattered light then goes through a second polarizer where it is detected and the resulting image is projected onto a screen.
  • the particles in the solution are being hit with the light and diffract the light in all directions.
  • the diffracted light from the particles can either interfere constructively (light regions) or destructively (dark regions). This process is repeated at short time intervals and the resulting set of speckle patterns are analyzed by an autocorrelator that compares the intensity of light at each spot over time.
  • SLS static light scattering
  • a high-intensity monochromatic light usually a laser, is launched in a solution containing the particles.
  • One or many detectors are used to measure the scattering intensity at one or many angles. The angular dependence is needed to obtain accurate measurements of both molar mass and size for all macromolecules of radius.
  • simultaneous measurements at several angles relative to the direction of incident light known as multi-angle light scattering (MALS) or multi-angle laser light scattering (MALLS) is generally regarded as the standard implementation of static light scattering.
  • MALS multi-angle light scattering
  • MALLS multi-angle laser light scattering
  • the particles described herein comprising an RNA payload i.e., RNA encoding a polypeptide comprising a cytokine or a functional variant thereof, to be delivered comprise a moiety incorporating into the particle, e.g., a hydrophobic moiety (e.g., lipid), having a binding moiety covalently attached thereto.
  • a moiety incorporating into the particle having a binding moiety covalently attached thereto is also referred to herein as "targeting compound”.
  • the moiety incorporating into the particle of the targeting compound relates to the part of the targeting compound that integrates into the particle comprising an RNA payload.
  • the binding moiety of the targeting compound relates to the part of the targeting compound that binds to target immune cells or forms the binding partner for a docking compound which binds to target immune cells.
  • the targeting compound is non-covalently incorporated into the particle comprising a payload, i.e., it forms an integral part of the particle, and the binding moiety of the targeting compound is covalently attached to a moiety incorporating into the particle in a manner such that it is available for binding to target immune cells or a docking compound.
  • the binding moiety of the targeting compound comprises a peptide or protein (e.g., an antibody or antibody fragment or a peptide tag).
  • the binding moiety of the targeting compound comprises a peptide or protein (e.g., an antibody or antibody fragment or a peptide tag) and is chemically linked, e.g., through a linker, to the moiety incorporating into the particle, e.g., hydrophobic moiety (e.g., lipid).
  • the targeting compound described herein comprises a hydrophobic component (e.g., lipid component) which allows it to be anchored in the particle.
  • the hydrophobic component comprises a moiety selected from vitamin E, dialkylamine, e.g., dimyristylamine (DMA), diacylglyceride, e.g., 1,2-dimyristoyl-sn-glycerol (DMG) and ceramide.
  • DMA dimyristylamine
  • DMG 1,2-dimyristoyl-sn-glycerol
  • the hydrophobic moiety comprises two C8-C24 hydrocarbon chains.
  • the hydrophobic moiety comprises two C10-C18 hydrocarbon chains.
  • the targeting compound described herein has as a hydrophobic group (e.g., lipid) a phospholipid, e.g., a biodegradable phospholipid such as phosphatidylethanolamine. In some embodiments, the targeting compound described herein has as a hydrophobic group (e.g., lipid) a glycerophospholipid.
  • the phospholipid is selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine), and mixtures thereof.
  • DSPE disearoylphosphatidylethanolamine
  • DPPE dipalmitoylphosphatidylethanolamine
  • DOPE dioleoylphosphatidylethanolamine
  • POPE palmitoyloleylphosphatidylethanolamine
  • the targeting compound comprises a polymer.
  • the moiety incorporating into the particle, e.g., hydrophobic moiety (e.g., lipid) of the targeting compound and the binding moiety of the targeting compound are connected through the polymer.
  • the polymer is a hydrophilic polymer.
  • the targeting compound comprises an amphiphilic derivative of the polymer.
  • the amphiphilic derivative of a polymer comprises a hydrophobic component (e.g., lipid component) which allows it to be anchored in the particle and a hydrophilic component of the polymer facing the outside of said particle, conferring hydrophilic properties at the surface thereof.
  • the amphiphilic derivatives of a polymer is inserted into the particle via its hydrophobic end. Consequently, the polymer component faces the outside of said particle and forms a protective hydrophilic shell surrounding the particle.
  • the polymer portion of the targeting compound contributes to conferring stealth properties on the particles.
  • the plasmatic half-life of the particles described herein is greater than 2 hours, e.g., between 3 and 10 hours. This characteristic advantageously allows the particles to accumulate at the target immune cells and to liberate therein their contents (payload) within reasonable amounts of time. The effectiveness of the targeted delivery described herein therefore increases as a result.
  • stealth is used herein to describe the ability of the particles described herein not to be detected and then sequestered and/or degraded, or to be hardly detected and then sequestered and/or degraded, and/or to be detected and then sequestered and/or degraded late, by the immune system of the host to which they are administered.
  • Macrophages constitute one of the most important components of the immune system and play a predominant role in eliminating foreign particles, including liposomes and other colloidal particles, from the blood circulation.
  • the clearance of particles takes place in two steps: opsonization by the depositing of serum proteins (or "opsonins") at the surface of the particles followed by recognition and capture of the opsonized particles by macrophages.
  • the polymer for use herein is selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) (including derivatives thereof).
  • PEG polyethylene glycol
  • pSar polysarcosine
  • POX polyoxazoline
  • POZ polyoxazine
  • pAEEA poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid
  • a polymer is designed to sterically stabilize a particle by forming a protective hydrophilic layer.
  • a polymer can reduce association of a particle with serum proteins and/or the resulting uptake by the reticuloendothelial system when such particles are administered in vivo.
  • the PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In some embodiments, the PEG is unsubstituted. In some embodiments, the PEG is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy or aryl groups.
  • the PEG has a molecular weight of from about 130 to about 50,000, in another embodiment about 150 to about 30,000, in another embodiment about 150 to about 20,000, in another embodiment about 150 to about 15,000, in another embodiment about 150 to about 10,000, in another embodiment about 150 to about 6000, in another embodiment about 150 to about 5000, in another embodiment about 150 to about 4000, in another embodiment about 150 to about 3000, in another embodiment about 300 to about 3000, in another embodiment about 1000 to about 3000, and in still another embodiment about 1500 to about 2500.
  • the PEG moiety of the targeting compound has a molecular weight of 1000 or more.
  • the PEG moiety of the targeting compound comprises 10 units or more of formula (O-CHz-CPhJn.
  • the PEG comprises from 20 to 200 ethylene oxide units, such as about 45 ethylene oxide units.
  • the PEG comprises "PEG2k”, also termed “PEG 2000”, which has an average molecular weight of about 2000 Daltons.
  • DSPE-PEG2000, DSPE-PEG3000 and DSPE-PEGsooo are used as the amphiphilic derivative of a polymer.
  • a pSar comprises between 2 and 200 sarcosine units, such as between 5 and 100 sarcosine units, between 10 and 50 sarcosine units, between 15 and 40 sarcosine units, e.g., about 23 sarcosine units.
  • a pSar comprises the structure of the following general formula: wherein s is the number of sarcosine units.
  • the POX and/or POZ polymer comprises between 2 and 200, between 2 and 190, between 2 and 180, between 2 and 170, between 2 and 160, between 2 and 150, between 2 and 140, between 2 and 130, between 2 and 120, between 2 and 110, between 2 and 100, between 2 and 90, between 2 and 80, between 2 and 70, between 5 and 200, between 5 and 190, between 5 and 180, between 5 and 170, between 5 and 160, between 5 and 150, between 5 and 140, between 5 and 130, between 5 and 120, between 5 and 110, between 5 and 100, between 5 and 90, between 5 and 80, between 5 and 70, between 10 and 200, between 10 and 190, between 10 and 180, between 10 and 170, between 10 and 160, between 10 and 150, between 10 and 140, between 10 and 130, between 10 and 120, between 10 and 110, between 10 and 100, between 10 and 90, between 10 and 80, or between 10 and 70 POX and/or POZ repeating units.
  • the POX and/or POZ polymer comprises the following general formula: wherein a is an integer between 1 and 2; Rn is alkyl, in particular C1-3 alkyl, such as methyl, ethyl, iso-propyl, or n-propyl, and is independently selected for each repeating unit; and m refers to the number of POX and/or POZ repeating units.
  • the POX and/or POZ polymer is a polymer of POX and comprises repeating units of the following general formula:
  • the POX and/or POZ polymer is a polymer of POZ and comprises repeating units of the following general formula:
  • m (/.e., the number of repeating units in the polymer) preferably is between 2 and 190, such as between 2 and 180, between 2 and 170, between 2 and 160, between 2 and 150, between 2 and 140, between 2 and 130, between 2 and 120, between 2 and 110, between 2 and 100, between 2 and 90, between 2 and 80, between 2 and 70, between 5 and 200, between 5 and 190, between 5 and 180, between 5 and 170, between 5 and 160, between 5 and 150, between 5 and 140, between 5 and 130, between 5 and 120, between 5 and 110, between 5 and 100, between 5 and 90, between 5 and 80, between 5 and 70, between 10 and 200, between 10 and 190, between 10 and 180, between 10 and 170, between 10 and 160, between 10 and 150, between 10 and 140, between 10 and 130, between 10 and 120, between 10 and 110, between 10 and 100, between 10 and 90, between 10 and 80, or between 10 and 70.
  • 2 and 190 such as between 2 and 180, between 2 and 170
  • m is 2 to 180, such as 4 to 160, 6 to 140, 8 to 120 or 10 to 100, e.g., 20 to 80, 30 to 70, or 40 to 50.
  • the POX and/or POZ polymer is a copolymer comprising repeating units of the following general formulas: wherein the number of repeating units shown on the left in the copolymer is 1 to 199; the number of repeating units of formula on the right in the copolymer is 1 to 199; and the sum of the number of repeating units of formula on the left and the number of repeating units of formula on the right in the copolymer is 2 to 200.
  • the number of repeating units of formula on the left in the copolymer is 1 to 179, such as 1 to 159, 1 to 139, 1 to 119 or 1 to 99; the number of repeating units of formula on the right in the copolymer is 1 to 179, such as 1 to 159, 1 to 139, 1 to 119 or 1 to 99; and the sum of the number of repeating units of formula on the left and the number of repeating units of formula on the right in the copolymer is 2 to 180, such as 4 to 160, 6 to 140, 8 to 120 or 10 to 100, e.g., 20 to 80, 30 to 70, or 40 to 50.
  • R11 at each occurrence may be the same alkyl group (e.g., R11 may be methyl in each repeating unit).
  • R11 in at least one repeating unit differs from R11 in another repeating unit (e.g., for at least one repeating unit R11 is one specific alkyl (such as ethyl), and for at least one different repeating unit R11 is a different specific alkyl (such as methyl)).
  • each R11 may be selected from two different alkyl groups (such as methyl and ethyl) and not all R11 are the same alkyl.
  • R11 preferably is methyl or ethyl, more preferably methyl.
  • each R11 is methyl or each R11 is ethyl.
  • R11 is independently selected from methyl and ethyl for each repeating unit, wherein in at least one repeating unit R11 is methyl, and in at least one repeating unit R11 is ethyl.
  • the polymer comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof.
  • the polymer comprises the following general formula: wherein
  • X 2 and X 1 taken together are optionally substituted amide, optionally substituted thioamide or ester;
  • Y is -CH2-, -(CH 2 ) 2 -, or -(CH 2 ) 3 -; z is 2 to 24; and n is 1 to 100.
  • X 1 is -C(O)- and X 2 is -NR 1 -, wherein R 1 is hydrogen or Ci-s alkyl. In some embodiments, X 1 is -C(O)- and X 2 is -NR 1 -, wherein R 1 is hydrogen or methyl. In some embodiments, X 1 is -C(O)- and X 2 is -NR 1 -, wherein R 1 is hydrogen.
  • Y is -CH 2 - or -(CH 2 ) 2 -. In some embodiments, Y is -CH 2 -.
  • the polymer comprises the following general formula: R 1 is hydrogen or Ci-s alkyl; z is 2 to 24; and n is 1 to 100.
  • z is 2 to 10. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.
  • the polymer comprises the following general formula: wherein
  • R 1 is hydrogen or Ci-8 alkyl; and n is 1 to 100.
  • R 1 is hydrogen or methyl. In some embodiments, R 1 is hydrogen.
  • the polymer comprises the following general formula: wherein n is 1 to 100.
  • n is 5 to 50. In some embodiments, n is 5 to 25.
  • n is 7 to 14. In some embodiments, n is 10 to 25. In some embodiments, n is 14 to 17. In some embodiments, n is 8 or 14.
  • the molar proportion of the targeting compoundintegrated into the particles is between 0.5 and 20 mol% of the lipid molecules making up the particle, preferably between 1 and 10 mol%.
  • the targeting compound comprises the following general formula:
  • L comprises a moiety incorporating the targeting compound into the particle, e.g., a hydrophobic moiety (e.g., lipid) attached to a first end of the polymer;
  • a hydrophobic moiety e.g., lipid
  • B comprises a binding moiety attached to a second end of the polymer
  • XI is absent or a first linking moiety
  • X2 is absent or a second linking moiety.
  • XI comprises a carbonyl group.
  • L comprises a phosphatidylethanolamine which may be linked to P by an amide group.
  • X2 comprises the reaction product of a thiol or cysteine reactive group, e.g., a maleimide group, with a thiol or cysteine group of a compound comprising the binding moiety.
  • a thiol or cysteine reactive group e.g., a maleimide group
  • L comprises a lipid as described above. In some embodiments, L comprises DSPE (distearoylphosphatidylethanolamine), DPPE
  • P comprises a polymer as described above.
  • P comprises a polymer which provides stealth property, extends circulation half-life and/or reduces non-specific protein binding or cell adhesion.
  • P comprises a polymer selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) (including derivatives thereof).
  • P comprises polyethyleneglycol (PEG); e.g., PEG as described above.
  • L-Xl-P comprises an amphiphilic derivative of a polymer as described above.
  • the amphiphilic derivative of a polymer comprises a conjugate of disteroyl-glycero-phosphoethanolamine (DSPE) and a polymer, e.g., a polymer as described above.
  • the amphiphilic derivative of a polymer comprises a disteroyl- glycero-phosphoethanolamine-polyethyleneglycol-conjugate (DSPE-PEG).
  • the targeting compound is obtainable by reacting the thiol or cysteine reactive group of a reagent comprising an amphiphilic derivative of a polymer, e.g., a PEG reagent comprising a hydrophobic moiety (e.g., lipid), with a thiol or cysteine group of a compound comprising the binding moiety.
  • a reagent comprising an amphiphilic derivative of a polymer, e.g., a PEG reagent comprising a hydrophobic moiety (e.g., lipid)
  • the thiol or cysteine reactive group comprises a maleimide group.
  • the PEG reagent comprises DSPE-PEG-maleimide.
  • the compound comprising the binding moiety comprises the formula SH(CH2) n C(O)-B, wherein n ranges from 1 to 5 and B comprises the binding moiety. In some embodiments, n is 2.
  • the targeting compound comprises the reaction product of 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)] with a compound comprising the formula SH(CH2) n C(O)-B, wherein n ranges from 1 to 5 and B comprises the binding moiety. In some embodiments, n is 2.
  • the targeting compound comprises the following general formula: L-X1-P-X2-B wherein L, XI, P and B are as described above and X2 comprises a thiosuccinimide moiety.
  • the targeting compound comprises the following general formula: wherein B comprises the binding moiety.
  • B comprises a moiety comprising the structure -N-peptide-C(O)-NH2.
  • the targeting compound comprises the following general formula: wherein P, X2 and B are as described above and Ri and R2 independently comprise an alkyl moiety.
  • at least one, e.g., each alkyl moiety is straight or branched, preferably straight.
  • at least one, e.g., each alkyl moiety has at least 8 carbon atoms, e.g., 8 to 24 such as 10 to 18 carbon atoms.
  • each alkyl moiety is the alkyl moiety of a fatty acid alcohol, more preferably at least one, e.g., each alkyl moiety is the alkyl moiety of a fatty acid alcohol having at least 8 carbon atoms, e.g., 8 to 24 such as 10 to 18 carbon atoms.
  • alkyl moieties include -(CFhJivCHs (stearyl), - (CH2)ISCH3 (palmityl), and -(CfhhsCHs (myristyl).
  • R1R2N- in the above formula is 1,2-dimyristylamine, wherein both alkyl groups are -(CfhhaCHs (myristyl).
  • the polymer P comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof.
  • the polymer P comprises the following general formula: wherein n is 5 to 50, e.g., 5 to 25, e.g., 7 to 14, e.g., 10 to 25, e.g., 14 to 17. In some embodiments, n is 8 or 14. In some embodiments, n is 14.
  • Ri and R2 in the above formula are -(CHzhsCHa (myristyl) and the polymer P comprises the following general formula: wherein n is 14.
  • the targeting compound comprises the following general formula: wherein P, X2 and B are as described above and each of R ti and R t 2 is independently H or methyl. In some embodiments, R ti and Rt2 are both methyl. In some embodiments, Rti is methyl, and Rt2 is H. In some embodiments, Rti is H, and R t 2 is methyl. In some embodiments, Rti and Rt2 are both H.
  • the targeting compound comprises the following general formula: wherein P, X2 and B are as described above.
  • the polymer P in the above formulas comprises poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof.
  • the polymer P comprises the following general formula: wherein n is 5 to 50, e.g., 5 to 25, e.g., 7 to 14, e.g., 10 to 25, e.g., 14 to 17. In some embodiments, n is 8 or 14. In some embodiments, n is 8. In some embodiments, n is 14.
  • the targeting compound comprises the following general formula: wherein XI, P, X2 and B are as described above and Ri and R 2 independently comprise an acyl moiety.
  • at least one, e.g., each acyl moiety is straight or branched, preferably straight.
  • at least one, e.g., each acyl moiety has at least 8 carbon atoms, e.g., 8 to 24 such as 10 to 18 carbon atoms.
  • each acyl moiety is the acyl moiety of a fatty acid, more preferably at least one, e.g., each acyl moiety is the acyl moiety of a fatty acid having at least 8 carbon atoms, e.g., 8 to 24 such as 10 to 18 carbon atoms.
  • acyl moieties include CH 3 (CH 2 )i6C(O)- (stearoyl), CH 3 (CH 2 )i4C(O)- (palmitoyl), and CH3(CH 2 )I 2 C(O)- (myristoyl).
  • both acyl groups are CH 3 (CH 2 )I 6 C(O)- (stearoyl). In some embodiments, both acyl groups are CH 3 (CH 2 )I 2 C(O)- (myristoyl).
  • XI is absent or comprises -HPO 3 -(CH 2 ) n - NH-, wherein n is 1 to 5, e.g., 2.
  • the polymer P comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof.
  • the polymer P comprises the following general formula: wherein n is 5 to 50, e.g., 5 to 25, e.g., 7 to 14, e.g., 10 to 25, e.g., 14 to 17. In some embodiments, n is 8 or 14. In some embodiments, n is 8. In some embodiments, n is 14.
  • the polymer P comprises a pSar. In some embodiments, the polymer P comprises the following general formula: wherein s is 2 to 200, e.g., 5 to 100, e.g., 10 to 50, e.g., 15 to 40. In some embodiments, s is 20 or 23.
  • the targeting compound comprises the following general formula: wherein P, X2 and B are as described above and Ri and R2 independently comprise an acyl moiety.
  • at least one, e.g., each acyl moiety is straight or branched, preferably straight.
  • at least one, e.g., each acyl moiety has at least 8 carbon atoms, e.g., 8 to 24 such as 10 to 18 carbon atoms.
  • each acyl moiety is the acyl moiety of a fatty acid, more preferably at least one, e.g., each acyl moiety is the acyl moiety of a fatty acid having at least 8 carbon atoms, e.g., 8 to 24 such as 10 to 18 carbon atoms.
  • acyl moieties include CH 3 (CH 2 )i6C(O)- (stearoyl), CH 3 (CH 2 )I 4 C(O)- (palmitoyl), and CH 3 (CH 2 )i2C(O)- (myristoyl).
  • both acyl groups are CH 3 (CH 2 )ieC(O)- (stearoyl).
  • both acyl groups are CH 3 (CH 2 )I 2 C(O)- (myristoyl).
  • the polymer P comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof.
  • the polymer P comprises the following general formula: wherein n is 5 to 50, e.g., 5 to 25, e.g., 7 to 14, e.g., 10 to 25, e.g., 14 to 17. In some embodiments, n is 8 or 14. In some embodiments, n is 8. In some embodiments, n is 14.
  • n 8 and Ri and R2 are CH3(CH2)ieC(O)- (stearoyl). In some embodiments, n is 14 and Ri and R2 are CH3(CH2)ieC(O)- (stearoyl).
  • n 8 and Ri and R2 are CH3(CH2)i2C(O)- (myristoyl). In some embodiments, n is 14 and Ri and R2 are CH3(CH2)i2C(O)- (myristoyl).
  • the polymer P comprises a pSar. In some embodiments, the polymer P comprises the following general formula: wherein s is 2 to 200, e.g., 5 to 100, e.g., 10 to 50, e.g., 15 to 40. In some embodiments, s is 20 or 23.
  • s is 20 and Ri and Fbare CH3(CH2)i6C(O)- (stearoyl).
  • s is 20 and Ri and Rzare CH3(CH2)i2C(O)- (myristoyl).
  • X2 in the above formulas comprises the reaction product of a thiol or cysteine reactive group, e.g., a maleimide group, with a compound comprising a thiol or cysteine group.
  • the compound comprising a thiol or cysteine group comprises the formula SH(CH2) n C(O)-, wherein n ranges from 1 to 5.
  • n is 2.
  • X2 comprises a thiosuccinimide moiety.
  • X2 comprises the following general formula: wherein nl and n2 are independently 1 to 5. In some embodiments, nl is 1 and n2 is 2. In some embodiments, nl is 2 and n2 is 1.
  • the present disclosure provides in one aspect, a targeting compound as described herein.
  • the binding moiety comprises a moiety binding to a cell surface antigen, e.g., a primary targeting moiety described herein.
  • the binding moiety comprises a moiety binding to a docking compound.
  • the binding moiety comprises a tag such as an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein. Accordingly, the present disclosure provides in one aspect, a compound the following general formula:
  • P comprises a polymer
  • L comprises a moiety incorporating the compound into the particle, e.g., a hydrophobic moiety (e.g., lipid) attached to a first end of the polymer;
  • a hydrophobic moiety e.g., lipid
  • B comprises a primary targeting moiety described herein, attached to a second end of the polymer
  • XI is absent or a first linking moiety
  • X2 is absent or a second linking moiety.
  • the present disclosure further provides in one aspect, a compound the following general formula:
  • P comprises a polymer
  • L comprises a moiety incorporating the compound into the particle, e.g., a hydrophobic moiety (e.g., lipid) attached to a first end of the polymer;
  • a hydrophobic moiety e.g., lipid
  • B comprises a tag such as an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein, attached to a second end of the polymer;
  • a tag such as an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein, attached to a second end of the polymer;
  • XI is absent or a first linking moiety
  • X2 is absent or a second linking moiety.
  • XI comprises a carbonyl group.
  • L comprises a phosphatidylethanolamine which may be linked to P by an amide group.
  • X2 comprises the reaction product of a thiol or cysteine reactive group, e.g., a maleimide group, with a thiol or cysteine group of a compound comprising the epitope tag. In some embodiments, X2 comprises a thiosuccinimide moiety.
  • L comprises a lipid as described above. In some embodiments, L comprises DSPE (distearoylphosphatidylethanolamine), DPPE
  • P comprises a polymer as described above.
  • P comprises a polymer which provides stealth property, extends circulation half-life and/or reduces non-specific protein binding or cell adhesion.
  • P comprises a polymer selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) (including derivatives thereof).
  • P comprises polyethyleneglycol (PEG); e.g., PEG as described above.
  • L-Xl-P comprises an amphiphilic derivative of a polymer as described above.
  • the amphiphilic derivative of a polymer comprises a conjugate of disteroyl-glycero-phosphoethanolamine (DSPE) and a polymer, e.g., a polymer as described above.
  • the amphiphilic derivative of a polymer comprises a disteroyl- glycero-phosphoethanolamine-polyethyleneglycol-conjugate (DSPE-PEG).
  • the targeting compound is obtainable by reacting the thiol or cysteine reactive group of a reagent comprising an amphiphilic derivative of a polymer, e.g., a PEG reagent comprising a hydrophobic moiety (e.g., lipid), with a thiol or cysteine group of a compound comprising the primary targeting moiety or epitope tag.
  • a reagent comprising an amphiphilic derivative of a polymer, e.g., a PEG reagent comprising a hydrophobic moiety (e.g., lipid)
  • the thiol or cysteine reactive group comprises a maleimide group.
  • the PEG reagent comprises DSPE-PEG-maleimide.
  • the compound comprising the primary targeting moiety or epitope tag comprises the formula SH(CH2) n C(O)-B, wherein n ranges from 1 to 5 and B comprises the primary targeting moiety or epitope tag.
  • n is 2.
  • the targeting compound comprises the reaction product of 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)] with a compound comprising the formula SH(CH2) n C(O)-B, wherein n ranges from 1 to 5 and B comprises the primary targeting moiety or epitope tag.
  • n is 2.
  • the targeting compound comprises the following general formula: wherein B comprises an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein.
  • the targeting compound comprises the following general formula: wherein X2 is as described above, Ri and R2 are CH3(CH2)ieC(O)- (stearoyl) or CH3(CH2)i2C(O)- (myristoyl), polymer P comprises the following general formula: wherein n is 5 to 50, e.g., 5 to 25, e.g., 7 to 14, e.g., 10 to 25, e.g., 14 to 17, e.g., 8 or 14, and B comprises an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein.
  • an ALFA-tag such as an ALFA-tag described herein.
  • n 8 and Ri and R2 are CH3(CH2)ieC(O)- (stearoyl). In some embodiments, n is 14 and Ri and R2 are CH3(CH2)i6C(O)- (stearoyl).
  • n 8 and Ri and R2 are CHs(CH2)i2C(O)- (myristoyl). In some embodiments, n is 14 and Ri and R2are CH3(CH2)i2C(O)- (myristoyl).
  • X2 comprises the following general formula:
  • the targeting compound comprises the following general formula: wherein X2 is as described above, Ri and R2 are CH3(CH2)ieC(O)- (stearoyl) or CH3(CH2)i2C(O)- (myristoyl), polymer P comprises the following general formula: wherein s is 2 to 200, e.g., 5 to 100, e.g., 10 to 50, e.g., 15 to 40, e.g., 20 or 23, and B comprises an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein. In some embodiments, s is 20 and Ri and R2 are CHs(CH2)i6C(O)- (stearoyl).
  • s is 20 and Ri and R2 are CH3(CH2)i2C(O)- (myristoyl).
  • X2 comprises the following general formula:
  • B comprises a moiety comprising the structure -N-peptide-C(O)-NH2, wherein peptide comprises an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein.
  • an ALFA-tag such as an ALFA-tag described herein.
  • the present disclosure provides in one aspect, a targeting compound as described above which is integrated in a particle (e.g., a particle as described herein), e.g., via a hydrophobic component (e.g., lipid component) of the targeting compound.
  • a targeting compound as described above which is integrated in a particle (e.g., a particle as described herein), e.g., via a hydrophobic component (e.g., lipid component) of the targeting compound.
  • an RNA payload is delivered specifically to a target immune cell by providing a moiety that binds to a target on target immune cells, e.g., an antigen on target immune cells, thus targeting particles comprising the RNA payload to the target immune cells.
  • a target on target immune cells e.g., an antigen on target immune cells
  • the moiety that binds to a target on target immune cells is comprised by a compound (targeting compound) which is an integral part of a particle carrying the payload.
  • the targeting compound comprises a binding moiety that binds to target immune cells.
  • the moiety that binds to a target on target immune cells is comprised by a compound (docking compound) further comprising a moiety that binds to a targeting compound which is an integral part of a particle carrying the payload and comprising a moiety for binding to the docking compound.
  • the targeting compound itself preferably does not comprise a moiety that binds to a target on target immune cells. Rather, the targeting compound comprises a binding moiety that forms the binding partner for a docking compound which binds to target immune cells.
  • a primary target is a cell surface antigen on target immune cells.
  • a "primary targeting moiety" as used herein relates to the part of the targeting compound or docking compound which binds to a primary target, e.g., a cell surface antigen on target immune cells.
  • a primary target e.g., a cell surface antigen on target immune cells.
  • Such targeting moieties are typically moieties that have affinity for cell surface targets. These moieties can be any peptide or protein (e.g. antibodies or antibody fragments) binding to the primary target.
  • suitable primary targeting moieties for use herein include cell surface antigen binding moieties, such as antibodies, antibody fragments and DARPins.
  • Other examples of primary targeting moieties are peptides or proteins which bind to a receptor.
  • a primary targeting moiety preferably binds with high specificity and/or high affinity and the bond with the primary target is preferably stable within the body.
  • the primary targeting moiety of the targeting compound or docking compound can comprise compounds including but not limited to antibodies, antibody fragments, e.g. Fab2, Fab, scFV, VHH domains, and other proteins or peptides.
  • the primary target is a cell surface antigen such as a T cell antigen, e.g., CD3, such as CD3e, CD8 or CD4, and suitable primary targeting moieties include but are not limited to, peptides and polypeptides targeting the cell surface antigen, e.g., antibodies, antibody fragments and DARPins.
  • the primary target is a receptor and suitable primary targeting moieties include but are not limited to, the ligand of such a receptor or a part thereof which still binds to the receptor, e.g., a receptor binding peptide in the case of receptor binding protein ligands.
  • the primary target and primary targeting moiety are selected so as to result in the specific or increased targeting of certain cells. This can be achieved by selecting primary targets with cell-specific expression.
  • T cell antigens e.g., those described herein, may be expressed in T cells while they are not expressed or expressed in a lower amount in other cells.
  • a "docking compound” is used to form a connection between a primary target, e.g., a target immune cell or an antigen on target immune cells, and a targeting compound which is integrated into a particle comprising an RNA payload to be delivered to a target immune cell.
  • a connection between a primary target, e.g., a target immune cell or an antigen on target immune cells, and a docking compound is a non- covalent connection.
  • a connection between a docking compound and a targeting compound is a non-covalent or covalent connection.
  • the targeting compound comprises a binding moiety for binding to the docking compound which is covalently attached to a hydrophobic moiety (e.g., lipid).
  • a hydrophobic moiety e.g., lipid
  • the hydrophobic moiety forms part of said particle.
  • a docking compound comprises a "primary targeting moiety", e.g., a moiety targeting a cell surface antigen on target immune cells, that is capable of binding to the primary target of interest, e.g., a cell surface antigen on target immune cells.
  • a "primary targeting moiety” as used herein relates to the part of the docking compound which binds to a primary target.
  • the docking compound further comprises a group which serves as a binding partner for a respective binding moiety of a targeting compound.
  • the portion of the targeting compound comprising the moiety incorporating the targeting compound into the particle e.g., the hydrophobic moiety (e.g., lipid) (having a binding moiety for the docking compound covalently attached) integrates into a particle carrying a payload and thus forms a connection between the particle and the docking compound.
  • the moiety of the docking compound binding to the targeting compound and the primary targeting moiety are linked to each other, preferably by a covalent linkage.
  • the docking compound comprises a bispecific molecule, such as a bispecific polypeptide, e.g., a bispecific antibody.
  • the docking compound comprises a binding domain binding to a primary target and a binding domain binding to a targeting compound.
  • the docking compound comprises an antibody or antibody fragment binding to a primary target and an antibody or antibody fragment binding to a targeting compound.
  • at least one binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody.
  • each binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody.
  • At least one binding domain comprises a single-domain antibody such as a VHH.
  • each binding domain comprises a single-domain antibody such as a VHH.
  • one binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody and the other binding domain comprises a single-domain antibody such as a VHH.
  • the binding domain binding to a primary target comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody.
  • the binding domain binding to a primary target comprises a single-domain antibody such as a VHH.
  • the binding domain binding to a targeting compound comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody.
  • the binding domain binding to a targeting compound comprises a single-domain antibody such as a VHH.
  • the docking compound comprises a fusion protein which comprises a binding domain binding to a primary target and a binding domain binding to a targeting compound.
  • the docking compound comprises a single peptide chain.
  • the single peptide chain comprises a portion, e.g., antibody, antibody fragment or DARPin, binding to a primary target and a portion, e.g., antibody or antibody fragment, binding to a targeting compound.
  • the antibody fragments are VHH, scFv, or a mixture thereof.
  • the docking compound comprises one of the following structures (from N- to C-terminus):
  • VHH (a targeting compound)-optional linker-VHH (a primary target) VHH (a primary target)-optional linker-VHH (a targeting compound) VHH (a targeting compound)-optional linker-scFv (a primary target) scFv (a primary target)-optional linker-VHH (a targeting compound) VHH (a primary target)-optional linker-scFv (a targeting compound) scFv (a targeting compoundj-optional linker-VHH (a primary target) scFv (a targeting compound)-optional linker-scFv (a primary target) scFv (a primary target)-optional linker-scFv (a targeting compound)
  • the docking compound comprises a bispecific molecule, such as a bispecific polypeptide, e.g., a bispecific antibody, wherein one specificity binds to an epitope tag, e.g., an ALFA-tag and the other scpecificity binds to a primary target, e.g., a cell surface antigen on target immune cells.
  • a primary target e.g., a cell surface antigen on target immune cells.
  • the specificity which binds to an epitope tag is an antibody or antibody fragment such as an NbALFA-nanobody (NbALFA).
  • NbALFA NbALFA
  • the specificity which binds to a primary target is an antibody, antibody fragment or DARPin.
  • the moiety targeting a primary target of the docking compound is selected from the group consisting of an anti-primary target DARPin, an anti-primary target VHH and an anti-primary target scFv and/or the moiety binding to a targeting compound of the docking compound is an NbALFA-nanobody (NbALFA).
  • the docking compound has a structure selected from the group consisting of NbALFA x anti-primary target DARPin, NbALFA x anti-primary target VHH and NbALFA x antiprimary target scFv.
  • the primary target is a T cell antigen, e.g., CD3, such CD3e, CD4 or CD8.
  • the docking compound comprises a bispecific antibody comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti- CD3 VHH.
  • the docking compound comprises a bispecific antibody comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-CD3 scFv.
  • the docking compound comprises a bispecific molecule comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-CD3 DARPin.
  • the docking compound comprises a bispecific antibody comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-CD4 VHH.
  • the docking compound comprises a bispecific antibody comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-CD4 scFv.
  • the docking compound comprises a bispecific molecule comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-CD4 DARPin.
  • the docking compound comprises a bispecific antibody comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-CD8 VHH.
  • the docking compound comprises a bispecific antibody comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-CD8 scFv.
  • the docking compound comprises a bispecific molecule comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti- CD8 DARPin.
  • the moiety on the targeting compound and the moiety on the docking compound interacting which each other non-covalently bind to each other.
  • the moieties on the targeting compound and on the docking compound interacting which each other bind to each other under physiological conditions.
  • the moieties on the targeting compound and on the docking compound interacting which each other are antibody/antigen systems.
  • the moiety of the targeting compound binding to the docking compound comprises a peptide or protein, e.g., a peptide tag, and the moiety of the docking compound binding to the targeting compound comprises a binder, e.g., an antibody or antibody fragment, binding to the peptide or protein.
  • the moiety of the docking compound binding to the targeting compound comprises a peptide or protein, e.g., a peptide tag
  • the moiety of the targeting compound bindingtothe docking compound comprises a binder, e.g., an antibody or antibody fragment, binding to the peptide or protein.
  • the moieties on the targeting compound and on the docking compound interacting which each other comprise an epitope tag/binder system.
  • tag refers to a chemical moiety which can be bound by another moiety.
  • an “epitope tag” refers to a stretch of amino acids to which an antibody or proteinaceous molecule with antibody-like function can bind.
  • the epitope tag comprises an ALFA-tag. In some embodiments, the epitope tag/binder system comprises an ALFA-tag and an ALFA-specific single-domain antibody (sdAb), NbALFA-nanobody.
  • sdAb single-domain antibody
  • an ALFA-tag comprises the amino acid sequence
  • AAO is Pro or deleted
  • AA1 is Ser, Gly, Thr, or Pro
  • AA2 is Arg, Gly, Ala, Glu, or Pro
  • AA3 is Leu, He, or Vai
  • AA4 is Glu or Gin
  • AA5 is Glu or Gin
  • AA6 is Glu or Gin
  • AA7 is Leu, He, or Vai
  • AA8 is Arg, Ala, Gin, or Glu
  • AA9 is Arg, Ala, Gin, or Glu
  • AA10 is Arg
  • AA11 is Leu
  • AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;
  • AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted.
  • AA14 is Pro or deleted.
  • an ALFA-tag comprises a sequence selected from the group consisting of SRLEEELRRRLTE, PSRLEEELRRRLTE, SRLEEELRRRLTEP, and PSRLEEELRRRLTEP.
  • an ALFA-tag comprises the cyclized amino acid sequence -AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12-AA13-AA14-, wherein the side-chains of any two of the amino acids of AAO, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13 and AA14 (XI, X2) are connected covalently; and wherein the amino acids of AAO, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13 and AA14
  • AAO is Pro or deleted
  • AA1 is Ser, Gly, Thr, or Pro
  • AA2 is Arg, Gly, Ala, Glu, or Pro
  • AA3 is Leu, lie, or Vai
  • AA4 is Glu or Gin
  • AA5 is Glu or Gin
  • AA6 is Glu or Gin
  • AA7 is Leu, lie, or Vai
  • AA8 is Arg, Ala, Gin, or Glu
  • AA9 is Arg, Ala, Gin, or Glu
  • AA10 is Arg
  • AA11 is Leu
  • AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;
  • AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted.
  • AA14 is Pro or deleted.
  • XI and X2 are separated by 2 or 3 amino acids.
  • AA5 is XI and AA9 is X2, AA5 is XI and AA8 is X2, AA9 is XI and AA13 is X2, AA6 is XI and AA9 is X2, AA9 is XI and AA12 is X2, AA10 is XI and AA13 is X2, AA6 is XI and AA10 is X2 or AA4 is XI and AA8 is X2.
  • an ALFA-tag comprises a cyclized amino acid sequence selected from the group consisting of a. -AA0-AAl-AA2-AA3-AA4-cyclo(Xl-AA6-AA7-AA8-X2)-Arg-Leu-AA12-AA13-AA14-, b. -AA0-AAl-AA2-AA3-AA4-cyclo(Xl-AA6-AA7-X2)-AA9-Arg-Leu-AA12-AA13-AA14-, c. -AA0-AAl-AA2-AA3-AA4-AA5-AA6-AA7-AA8-cyclo(Xl-Arg-Leu-AA12-X2)-AA14-, d.
  • AAO is Pro or deleted
  • AA1 is Ser, Gly, Thr, or Pro
  • AA2 is Arg, Gly, Ala, Glu, or Pro
  • AA3 is Leu, He, or Vai
  • AA4 is Glu or Gin
  • AA5 is Glu or Gin
  • AA6 is Glu or Gin
  • AA7 is Leu, He, or Vai
  • AA8 is Arg, Ala, Gin, or Glu
  • AA9 is Arg, Ala, Gin, or Glu
  • AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;
  • AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted.
  • AA14 is Pro or deleted.
  • Xi and X2 in the peptides disclosed herein are connected covalently via an amide, disulfide, thioether, ether, ester, thioester, thioamide, alkylene, alkenylene, alkynylene, and/or 1,2,3-triazole.
  • a cyclized amino acid sequence described herein is generated by linking an amino group of a side-chain of one of Xi and X2 to the carboxyl group of a side-chain of the other of Xi and X2 via an amide bond.
  • the amino group of the side chain of an amino acid that possesses a pendant amine group, e.g., lysine or a lysine derivative, and the carboxyl group of the side chain of an acidic amino acid, e.g., aspartic acid, glutamic acid or a derivative thereof, can be used to generate a cyclized amino acid sequence via an amide bond.
  • a cyclized amino acid sequence described herein is generated by linking a sulfhydryl group of a side-chain of one of Xi and X2 to the sulfhydryl group of a side- chain of the other of Xi and X2 via a disulfide bond.
  • Sulfhydryl group-containing amino acids include cysteine and other sulfhydryl-containing amino acids as Pen.
  • Xi and X2 are, independently, selected from the group consisting of Glu, DGIu, Asp, DAsp, Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, DDap, Cys, DCys, hCys, DhCys, Pen, and DPen, with the proviso that when Xi is Glu, DGIu, Asp, or DAsp, X2 is Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, or DDap; when XI is Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, or DDap, X2 is Glu, DGIu, Asp, or DAsp; and when X
  • Xi is Glu and X2 is Lys.
  • -cyclo(Glu - Lys)-, - c(Glu - Lys)-, -cyclo(E - K)-, -c(E - K)-, -E K- cyclo, or -cycloE — cycloK- comprises the following structure:
  • Xi is Lys and X2 is Glu.
  • -cyclo(Lys - Glu)-, - c(Lys - Glu)-, -cyclo(K - E)-, -c(K - E)-, -K - E- cyclo, or cycloK - cycloE- comprises the following structure:
  • Xi is Cys and X2 is Cys.
  • -cyclo(Cys - Cys)-, c(Cys - Cys)-, -cyclo(C - C)-, -c(C - C)-, -C — C- cyclo, or -cycloC - cycloC- comprises the following structure:
  • the cyclic peptide is attached to a 3-mercaptopropionyl moiety through an a-amine moiety of the leftmost amino acid in the cyclic peptide.
  • the rightmost amino acid in the cyclic peptide comprises an amide.
  • the cyclized amino acid sequence is one selected from the group consisting of
  • the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Glu-Glu- Leu-Arg-Lys)-Arg-Leu-Thr-Glu-. In some other embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-. In yet some other embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Lys)- Arg-Arg-Leu-Thr-Glu-. In still some other embodiments, the cyclized amino acid sequence is - Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Glu)-.
  • the cyclic peptides may have different cyclic bridging moieties forming the ring structure.
  • chemically stable bridging moieties are included in the ring structure such as, for example, an amide group, a lactone group, an ether group, a thioether group, a disulfide group, an alkylene group, an alkenyl group, or a 1,2,3-triazole.
  • an ALFA-tag binding moiety comprises an antibody or antibody fragment, e.g., a camelid VHH domain.
  • an ALFA-tag binding moiety comprises a single-domain antibody (sdAb), NbALFA-nanobody.
  • an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence VTXiSALNAMAMG, wherein Xi is I or V, the CDR2 sequence AVSX2RGNAM, wherein X2 is E, H, N, D, or S, and the CDR3 sequence LEDRVDSFHDY.
  • a camelid VHH domain comprising the CDR1 sequence VTXiSALNAMAMG, wherein Xi is I or V, the CDR2 sequence AVSX2RGNAM, wherein X2 is E, H, N, D, or S, and the CDR3 sequence LEDRVDSFHDY.
  • an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence GVTXiSALNAMAMG, wherein Xi is I or V, the CDR2 sequence AVSX2RGNAM, wherein X2 is E, H, N, D, or S, and the CDR3 sequence LEDRVDSFHDY.
  • a camelid VHH domain comprising the CDR1 sequence GVTXiSALNAMAMG, wherein Xi is I or V, the CDR2 sequence AVSX2RGNAM, wherein X2 is E, H, N, D, or S, and the CDR3 sequence LEDRVDSFHDY.
  • an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence VTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY.
  • an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence GVTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY.
  • an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the amino acid sequence EVQLQESGGGLVQ.PGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSERGNAMYRESV QGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence, or a fragment of said amino acid sequence or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence.
  • the amino acid sequence comprises CDR1, CDR2 and CDR3 sequences as described above.
  • the epitope tag/binder system comprises an epitope tag comprising the sequence PDRVRAVSHWSS (Spot-tag) and the binder comprises a single-domain antibody (sdAb, or nanobody) (Spot-nanobody (14.7 kD)) that specifically binds to the Spot-tag.
  • sdAb single-domain antibody
  • spot-nanobody (14.7 kD)
  • the system used herein may comprise a Tag/Catcher system forming a covalent bond, e.g., SpyTag/SpyCatcher forming an isopeptide bond.
  • the SpyTag/SpyCatcher system is a technology for irreversible conjugation of recombinant proteins.
  • the peptide SpyTag spontaneously reacts with the protein SpyCatcher to form an intermolecular isopeptide bond between the pair.
  • bioconjugation can be achieved between two recombinant proteins.
  • the present disclosure provides in one aspect, a complex wherein a particle comprising a targeting compound is bound to a docking compound.
  • the targeting compound and the docking compound comprise moieties interacting which each other.
  • the targeting compound comprises an ALFA-tag.
  • the moiety binding to a targeting compound of the docking compound may be a NbALFA-nanobody (NbALFA).
  • the docking compound may have a structure selected from the group consisting of NbALFA x anti-primary target DARPin, NbALFA x anti-primary target VHH and NbALFA x anti-primary target scFv.
  • the targeting compound described herein is a lipid-PEG-peptide conjugate compound having the structure (I): wherein the Lipid is a phospholipid attached to the carbonyl through the amino group of the ethanolamine moiety thereof, PEG has a molecular weight of from about 130 to about 50,000, a-amino group of the left most amino acid group of the Peptide is attached to the carbonyl group of the 3-mercaptopropionyl moiety and the Peptide comprises from about 11 to about 15 amino acids, and Z is a bond or -CH2-.
  • the lipid-PEG-peptide conjugate has the following structure (II) k particular embodiment of the lipid-PEG-peptide conjugate compound having the structure (I) or (II) is where Z is a bond.
  • the PEPTIDE has the sequence -SRLEEELRRRLTE-. In another embodiment, the PEPTIDE has the sequence -PSRLEEELRRRLTE-.
  • the PEPTIDE is a cyclic peptide selected from the group consisting of -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Ser-Arg- Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu- Lys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Glu)-, - Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Asp- Glu-Leu-Lys)-Arg-Arg-Arg-Arg
  • PEG as used in the above formula I means any polyethylene glycol or other polyalkylene ether polymer.
  • PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide.
  • PEG is unsubstituted.
  • the PEG is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy or aryl groups.
  • the PEG has a molecular weight of from about 130 to about 50,000, in another embodiment about 150 to about 30,000, in another embodiment about 150 to about 20,000, in another embodiment about 150 to about 15,000, in another embodiment about 150 to about 10,000, in another embodiment about 150 to about 6000, in another embodiment about 150 to about 5000, in another embodiment about 150 to about 4000, in another embodiment about 150 to about 3000, in another embodiment about 300 to about 3000, in another embodiment about 1000 to about 3000, and in still another embodiment about 1500 to about 2500.
  • the PEG conjuggated to the lipid
  • PEG2k also termed "PEG 2000” which has an average molecular weight of about 2000 Daltons.
  • PEG 2000 polyethylene glycol PEG
  • pegylated lipid is another name for a lipid comprising polyethylene glycol PEG, such as, for example PEG2K, and, if a phosphorous-containing linkage is present, the lipid is generally referred to herein as a “phospholipid” or a “pegylated phospholipid.”
  • the lipid portion to which the PEG is bonded in the functionalized stealth lipid disclosed in the above formula I comprises a neutral phospholipid.
  • neutral phospholipids include, but are not limited to: dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoyl-sn-glycero-3-phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn- glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1- myristoyl-2-palmitoyl phosphatidylcholine (MPPC), l-palmitoyl
  • PEPTIDE or “Peptide” are used interchangeably and as used in the above formula I or II refers to a series of amino acids connected one to another by peptide bonds between the amino and carboxy groups of adjacent residues.
  • PEPTIDE-NH2 represents that the C- terminus carboxyl group of the peptide is an amide.
  • the term “PEPTIDE” or “Peptide” refers to an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein.
  • the particles described herein comprise one or more particle forming agents, an RNA payload to be delivered to a target immune cell and a targeting compound for binding to the target immune cells or the docking compound.
  • an RNA payload is delivered to target immune cells to transfect the target immune cells and enable the target immune cells to express a cytokine or a functional variant thereof encoded by the RNA.
  • cytokines examples include interferons, such as interferon-alpha (IFN-a) or interferongamma (IFN-y), interleukins, such as IL-2, IL- 7, IL-12, IL-15 and IL-21, colony stimulating factors, such as M-CSF and GM-CSF, and tumor necrosis factor.
  • interferons such as interferon-alpha (IFN-a) or interferongamma (IFN-y)
  • interleukins such as IL-2, IL- 7, IL-12, IL-15 and IL-21
  • colony stimulating factors such as M-CSF and GM-CSF
  • tumor necrosis factor examples include interferons, such as interferon-alpha (IFN-a) or interferongamma (IFN-y), interleukins, such as IL-2, IL- 7, IL-12, IL-15 and IL-21, colony stimulating factors, such as M-CSF and GM-CSF,
  • the cytokine is involved in and preferably induces or enhances development, priming, expansion, differentiation and/or survival of T cells.
  • the cytokine is an interleukin.
  • the cytokine is an interleukin selected from the group consisting of IL-2, IL-7, IL-12, IL-15, and IL-21.
  • cytokines relates to proteins which have a molecular weight of about 5 to 60 kDa and which participate in cell signaling (e.g., paracrine, endocrine, and/or autocrine signaling). In particular, when released, cytokines exert an effect on the behavior of cells around the place of their release. Examples of cytokines include lymphokines, interleukins, chemokines, interferons, and tumor necrosis factors (TNFs). According to the present disclosure, cytokines do not include hormones or growth factors.
  • Cytokines differ from hormones in that (i) they usually act at much more variable concentrations than hormones and (ii) generally are made by a broad range of cells (nearly all nucleated cells can produce cytokines).
  • cytokines include erythropoietin (EPO), colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF), bone morphogenetic protein (BMP), interferon alfa (IFN-a), interferon beta (IFN-p), interferon gamma (INF-y), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 10 (IL-10), interleukin 11 (IL-11), interleukin 12 (IL-12), interleukin 15 (IL-15), and interleukin 21 (IL-21), as well as variants and derivatives thereof.
  • EPO erythrop
  • a cytokine may be a naturally occurring cytokine or a functional fragment or variant thereof.
  • a cytokine may be human cytokine and may be derived from any vertebrate, especially any mammal.
  • One particularly preferred cytokine is IL-2 or a functional fragment or variant thereof.
  • a suitable cytokine for use herein is a cytokine involved in T cell proliferation and/or maintenance.
  • suitable cytokines include IL-2 or IL-7, fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants, such as extended-PK cytokines.
  • a suitable cytokine for use herein is a cytokine involved in inducing an immune response, in particular in T-cell priming or activation of resident immune cells.
  • cytokines involved in T cell priming include IL-12, IL-15, IFN-a, or IFN-0, fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants, such as extended-PK cytokines.
  • Interferons are a group of signaling proteins made and released by host cells in response to the presence of several pathogens, such as viruses, bacteria, parasites, and also tumor cells. In a typical scenario, a virus-infected cell will release interferons causing nearby cells to heighten their anti-viral defenses. Interferons are usually characterized by antiviral, antiproliferative and immunomodulatory activities. Interferons are proteins that alter and regulate the transcription of genes within a cell by binding to interferon receptors on the regulated cell's surface, thereby preventing viral replication within the cells. Based on the type of receptor through which they signal, interferons are typically divided among three classes: type I interferon, type II interferon, and type III interferon.
  • IFNAR IFN-a/P receptor
  • IFNa The type I interferons present in humans are IFNa, IFNp, IFNe, IFNK and IFNw.
  • type I interferons present in humans are IFNa, IFNp, IFNe, IFNK and IFNw.
  • type I interferons present in humans are IFNa, IFNp, IFNe, IFNK and IFNw.
  • type I interferons present in humans are IFNa, IFNp, IFNe, IFNK and IFNw.
  • type IFNa In general, type IFNa, IFNp, IFNe, IFNK and IFNw.
  • I interferons are produced when the body recognizes a virus that has invaded it. They are produced by fibroblasts and monocytes. Once released, type I interferons bind to specific receptors on target cells, which leads to expression of proteins that will prevent the virus from producing and replicating its RNA and DNA.
  • the IFN-a proteins are produced mainly by plasmacytoid dendritic cells (pDCs). They are mainly involved in innate immunity against viral infection.
  • the genes responsible for their synthesis come in 13 subtypes that are called IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21. These genes are found together in a cluster on chromosome 9.
  • the IFN-P proteins are produced in large quantities by fibroblasts. They have antiviral activity that is involved mainly in innate immune response. Two types of IFN-P have been described, IFN-pl and IFN-p3. The natural and recombinant forms of IFN-pl have antiviral, antibacterial, and anticancer properties.
  • Type II interferon IFN-y in humans
  • I L12 Type II interferon
  • type II interferons are released by cytotoxic T cells and T helper cells.
  • Type III interferons signal through a receptor complex consisting of IL1OR2 (also called CRF2- 4) and IFNLR1 (also called CRF2-12). Although discovered more recently than type I and type
  • type III IFNs in some types of virus or fungal infections.
  • type I and II interferons are responsible for regulating and activating the immune response.
  • a type I interferon is preferably IFN-a or IFN-P, more preferably IFN-a.
  • an interferon may be a naturally occurring interferon or a functional fragment or variant thereof.
  • An interferon may be human interferon and may be derived from any vertebrate, especially any mammal.
  • Interleukins are a group of cytokines (secreted proteins and signal molecules) that can be divided into four major groups based on distinguishing structural features. However, their amino acid sequence similarity is rather weak (typically 15-25% identity). The human genome encodes more than 50 interleukins and related proteins.
  • an interleukin may be a naturally occurring interleukin or a functional fragment or variant thereof.
  • An interleukin may be human interleukin and may be derived from any vertebrate, especially any mammal.
  • Cytokine polypeptides described herein can be prepared as fusion or chimeric polypeptides that include a portion comprising a cytokine or a functional variant thereof and a heterologous polypeptide (i.e., a polypeptide that is not a cytokine or a functional variant thereof).
  • a cytokine or functional variant thereof may be fused to an extended-PK group, which increases circulation half-life.
  • extended-PK groups are described infra. It should be understood that other PK groups that increase the circulation half-life of cytokines, or variants thereof, are also applicable to the present disclosure.
  • the extended-PK group is a serum albumin domain (e.g., mouse serum albumin, human serum albumin).
  • PK is an acronym for "pharmacokinetic” and encompasses properties of a compound including, by way of example, absorption, distribution, metabolism, and elimination by a subject.
  • an "extended-PK group” refers to a protein, peptide, or moiety that increases the circulation half-life of a biologically active molecule when fused to or administered together with the biologically active molecule.
  • examples of an extended-PK group include serum albumin (e.g., HSA), Immunoglobulin Fc or Fc fragments and variants thereof, transferrin and variants thereof, and human serum albumin (HSA) binders (as disclosed in U.S. Publication Nos. 2005/0287153 and 2007/0003549).
  • extended-PK groups are disclosed in Kontermann, Expert Opin Biol Ther, 2016 Jul;16(7) :903- 15 which is herein incorporated by reference in its entirety.
  • an "extended-PK" cytokine refers to a cytokine moiety (including functional variants thereof) in combination with an extended-PK group.
  • the extended-PK cytokine is a fusion protein in which a cytokine moiety is linked or fused to an extended-PK group.
  • the serum half-life of an extended-PK cytokine is increased relative to the cytokine alone (i.e., the cytokine not fused to an extended-PK group). In certain embodiments, the serum half-life of the extended-PK cytokine is at least 20%, at least 40%, at least 60%, at least 80%, at least 100%, at least 120%, at least 150%, at least 180%, at least 200%, at least 400%, at least 600%, at least 800%, or at least 1000% longer relative to the serum half-life of the cytokine alone.
  • the serum half-life of the extended-PK cytokine is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5- fold, 6-fold, 7-fold, 8-fold, 10-fold, 12-fold, 13-fold, 15-fold, 17-fold, 20-fold, 22-fold, 25-fold, 27-fold, 30-fold, 35-fold, 40-fold, or 50-fold greater than the serum half-life of the cytokine alone.
  • the serum half-life of the extended-PK cytokine is at least 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200 hours.
  • half-life refers to the time taken for the serum or plasma concentration of a compound such as a peptide or polypeptide to reduce by 50%, in vivo, for example due to degradation and/or clearance or sequestration by natural mechanisms.
  • An extended-PK cytokine suitable for use herein is stabilized in vivo and its half-life increased by, e.g., fusion to serum albumin (e.g., HSA or MSA), which resist degradation and/or clearance or sequestration.
  • the half-life can be determined in any manner known per se, such as by pharmacokinetic analysis.
  • Suitable techniques will be clear to the person skilled in the art, and may for example generally involve the steps of suitably administering a suitable dose of the amino acid sequence or compound to a subject; collecting blood samples or other samples from said subject at regular intervals; determining the level or concentration of the amino acid sequence or compound in said blood sample; and calculating, from (a plot of) the data thus obtained, the time until the level or concentration of the amino acid sequence or compound has been reduced by 50% compared to the initial level upon dosing. Further details are provided in, e.g., standard handbooks, such as Kenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al., Pharmacokinetic Analysis: A Practical Approach (1996). Reference is also made to Gibaldi, M. et al., Pharmacokinetics, 2nd Rev. Edition, Marcel Dekker (1982).
  • the extended-PK group includes serum albumin, or fragments thereof or variants of the serum albumin or fragments thereof (all of which for the purpose of the present disclosure are comprised by the term "albumin”).
  • Polypeptides described herein may be fused to albumin (or a fragment or variant thereof) to form albumin fusion proteins.
  • albumin fusion proteins are described in U.S. Publication No. 20070048282.
  • albumin fusion protein refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) to at least one molecule of a protein such as a therapeutic protein, in particular a cytokine.
  • the albumin fusion protein may be generated by translation of a nucleic acid in which a polynucleotide encoding a therapeutic protein is joined in-frame with a polynucleotide encoding an albumin.
  • an albumin fusion protein comprises at least one molecule of a therapeutic protein (including, but not limited to a mature form of the therapeutic protein) and at least one molecule of albumin (including but not limited to a mature form of albumin).
  • an albumin fusion protein is processed by a host cell such as a cell of the target organ for administered RNA, and secreted into the circulation.
  • Processing of the nascent albumin fusion protein that occurs in the secretory pathways of the host cell used for expression of the RNA may include, but is not limited to signal peptide cleavage; formation of disulfide bonds; proper folding; addition and processing of carbohydrates (such as for example, N- and O-linked glycosylation); specific proteolytic cleavages; and/or assembly into multimeric proteins.
  • An albumin fusion protein is preferably encoded by RNA in a non-processed form which in particular has a signal peptide at its N-terminus and following secretion by a cell is preferably present in the processed form wherein in particular the signal peptide has been cleaved off.
  • albumin fusion protein refers to an albumin fusion protein product which has undergone N-terminal signal peptide cleavage, herein also referred to as a "mature albumin fusion protein".
  • albumin fusion proteins comprising a therapeutic protein have a higher plasma stability compared to the plasma stability of the same therapeutic protein when not fused to albumin.
  • Plasma stability typically refers to the time period between when the therapeutic protein is administered in vivo and carried into the bloodstream and when the therapeutic protein is degraded and cleared from the bloodstream, into an organ, such as the kidney or liver, that ultimately clears the therapeutic protein from the body. Plasma stability is calculated in terms of the half-life of the therapeutic protein in the bloodstream. The halflife of the therapeutic protein in the bloodstream can be readily determined by common assays known in the art.
  • albumin refers collectively to albumin protein or amino acid sequence, or an albumin fragment or variant, having one or more functional activities (e.g., biological activities) of albumin.
  • albumin refers to human albumin or fragments or variants thereof especially the mature form of human albumin, or albumin from other vertebrates or fragments thereof, or variants of these molecules.
  • the albumin may be derived from any vertebrate, especially any mammal, for example human, cow, sheep, or pig. Nonmammalian albumins include, but are not limited to, hen and salmon.
  • the albumin portion of the albumin fusion protein may be from a different animal than the therapeutic protein portion.
  • the albumin is human serum albumin (HSA), or fragments or variants thereof, such as those disclosed in US 5,876,969, WO 2011/124718, WO 2013/075066, and WO 2011/0514789.
  • HSA human serum albumin
  • human serum albumin HSA
  • human albumin HA
  • albumin and serum albumin are broader, and encompass human serum albumin (and fragments and variants thereof) as well as albumin from other species (and fragments and variants thereof).
  • a fragment of albumin sufficient to prolong the therapeutic activity or plasma stability of the therapeutic protein refers to a fragment of albumin sufficient in length or structure to stabilize or prolong the therapeutic activity or plasma stability of the protein so that the plasma stability of the therapeutic protein portion of the albumin fusion protein is prolonged or extended compared to the plasma stability in the non-fusion state.
  • the albumin portion of the albumin fusion proteins may comprise the full length of the albumin sequence, or may include one or more fragments thereof that are capable of stabilizing or prolonging the therapeutic activity or plasma stability.
  • Such fragments may be of 10 or more amino acids in length or may include about 15, 20, 25, 30, 50, or more contiguous amino acids from the albumin sequence or may include part or all of specific domains of albumin.
  • one or more fragments of HSA spanning the first two immunoglobulin- like domains may be used.
  • the HSA fragment is the mature form of HSA.
  • an albumin fragment or variant will be at least 100 amino acids long, preferably at least 150 amino acids long.
  • albumin may be naturally occurring albumin or a fragment or variant thereof.
  • Albumin may be human albumin and may be derived from any vertebrate, especially any mammal.
  • the albumin fusion protein comprises albumin as the N-terminal portion, and a therapeutic protein as the C-terminal portion.
  • an albumin fusion protein comprising albumin as the C-terminal portion, and a therapeutic protein as the N-terminal portion may also be used.
  • the albumin fusion protein has a therapeutic protein fused to both the N-terminus and the C-terminus of albumin.
  • the therapeutic proteins fused at the N- and C-termini are the same therapeutic proteins.
  • the therapeutic proteins fused at the N- and C- termini are different therapeutic proteins.
  • the different therapeutic proteins are both cytokines.
  • the therapeutic protein(s) is (are) joined to the albumin through (a) peptide linker(s).
  • a peptide linker between the fused portions may provide greater physical separation between the moieties and thus maximize the accessibility of the therapeutic protein portion, for instance, for binding to its cognate receptor.
  • the peptide linker may consist of amino acids such that it is flexible or more rigid.
  • the linker sequence may be cleavable by a protease or chemically.
  • Fc region refers to the portion of a native immunoglobulin formed by the respective Fc domains (or Fc moieties) of its two heavy chains.
  • Fc domain refers to a portion or fragment of a single immunoglobulin (Ig) heavy chain wherein the Fc domain does not comprise an Fv domain.
  • an Fc domain begins in the hinge region just upstream of the papain cleavage site and ends at the C-terminus of the antibody. Accordingly, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain.
  • an Fc domain comprises at least one of: a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragment thereof.
  • a hinge e.g., upper, middle, and/or lower hinge region
  • a CH2 domain e.g., a CH2 domain, and a CH3 domain
  • an Fc domain comprises a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof).
  • an Fc domain comprises a CH2 domain (or portion thereof) fused to a CH3 domain (or portion thereof).
  • an Fc domain consists of a CH3 domain or portion thereof.
  • an Fc domain consists of a hinge domain (or portion thereof) and a CH3 domain (or portion thereof). In certain embodiments, an Fc domain consists of a CH2 domain (or portion thereof) and a CH3 domain. In certain embodiments, an Fc domain consists of a hinge domain (or portion thereof) and a CH2 domain (or portion thereof). In certain embodiments, an Fc domain lacks at least a portion of a CH2 domain (e.g., all or part of a CH2 domain).
  • An Fc domain herein generally refers to a polypeptide comprising all or part of the Fc domain of an immunoglobulin heavy-chain.
  • the Fc domain may be derived from an immunoglobulin of any species and/or any subtype, including, but not limited to, a human IgGl, lgG2, lgG3, lgG4, IgD, IgA, IgE, or IgM antibody.
  • the Fc domain encompasses native Fc and Fc variant molecules.
  • any Fc domain may be modified such that it varies in amino acid sequence from the native Fc domain of a naturally occurring immunoglobulin molecule.
  • the Fc domain has reduced effector function (e.g., FcyR binding).
  • an Fc domain of a polypeptide described herein may be derived from different immunoglobulin molecules.
  • an Fc domain of a polypeptide may comprise a CH2 and/or CH3 domain derived from an IgGl molecule and a hinge region derived from an lgG3 molecule.
  • an Fc domain can comprise a chimeric hinge region derived, in part, from an IgGl molecule and, in part, from an lgG3 molecule.
  • an Fc domain can comprise a chimeric hinge derived, in part, from an IgGl molecule and, in part, from an lgG4 molecule.
  • an extended-PK group includes an Fc domain or fragments thereof or variants of the Fc domain or fragments thereof (all of which for the purpose of the present disclosure are comprised by the term "Fc domain").
  • the Fc domain does not contain a variable region that binds to antigen.
  • Fc domains suitable for use in the present disclosure may be obtained from a number of different sources.
  • an Fc domain is derived from a human immunoglobulin.
  • the Fc domain is from a human IgGl constant region. It is understood, however, that the Fc domain may be derived from an immunoglobulin of another mammalian species, including for example, a rodent (e.g. a mouse, rat, rabbit, guinea pig) or non-human primate (e.g. chimpanzee, macaque) species.
  • rodent e.g. a mouse, rat, rabbit, guinea pig
  • non-human primate e.g. chimpanzee, mac
  • the Fc domain (or a fragment or variant thereof) may be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgGl, lgG2, lgG3, and lgG4.
  • Fc domain gene sequences e.g., mouse and human constant region gene sequences
  • Constant region domains comprising an Fc domain sequence can be selected lacking a particular effector function and/or with a particular modification to reduce immunogenicity.
  • Many sequences of antibodies and antibody-encoding genes have been published and suitable Fc domain sequences (e.g. hinge, CH2, and/or CH3 sequences, or fragments or variants thereof) can be derived from these sequences using art recognized techniques.
  • the extended-PK group is a serum albumin binding protein such as those described in US2005/0287153, US2007/0003549, US2007/0178082, US2007/0269422, US2010/0113339, W02009/083804, and W02009/133208, which are herein incorporated by reference in their entirety.
  • the extended-PK group is transferrin, as disclosed in US 7,176,278 and US 8,158,579, which are herein incorporated by reference in their entirety.
  • the extended-PK group is a serum immunoglobulin binding protein such as those disclosed in US2007/0178082, US2014/0220017, and US2017/0145062, which are herein incorporated by reference in their entirety.
  • the extended-PK group is a fibronectin (Fn)-based scaffold domain protein that binds to serum albumin, such as those disclosed in US2012/0094909, which is herein incorporated by reference in its entirety. Methods of making fibronectin-based scaffold domain proteins are also disclosed in US2012/0094909.
  • Fn3-based extended-PK group is Fn3(HSA), i.e., a Fn3 protein that binds to human serum albumin.
  • the extended-PK cytokine can employ one or more peptide linkers.
  • peptide linker refers to a peptide or polypeptide sequence which connects two or more domains (e.g., the extended- PK moiety and a cytokine moiety) in a linear amino acid sequence of a polypeptide chain.
  • peptide linkers may be used to connect a cytokine moiety to a HSA domain.
  • Linkers suitable for fusing the extended-PK group to, e.g., a cytokine are well known in the art.
  • Exemplary linkers include glycine-serine-polypeptide linkers, glycine-proline-polypeptide linkers, and proline-alanine polypeptide linkers.
  • the linker is a glycine-serine-polypeptide linker, i.e., a peptide that consists of glycine and serine residues.
  • IL-2 A particularly preferred cytokine for use herein is IL-2 or a functional variant thereof.
  • Human IL-2 is a key cytokine in T cell immunity. It supports the differentiation, proliferation, survival and effector functions of T cells (Gillis S, Smith KA, Nature 1977; 268(5616): 154-56, Blattman JN et aL, Nat Med 2003; 9(5): 540-47, Bamford RN et al., Proc Natl Acad Sci USA. 1994; 91(11): 4940-44, Kamimura D, Bevan MJ, J Exp Med 2007; 204(8): 1803-12).
  • rlL-2 aldesleukin
  • Recombinant rlL-2 was the first approved cancer immunotherapy and has been used for decades in the treatment of late stage malignant melanoma and renal cell cancer (Kammula US et al., Cancer 1998; 83(4): 797-805). Most patients with complete responses after rlL-2 treatment remain regression free for more than 25 years after initial treatment, but overall response rates are low (Klapper JA et al., Cancer 2008; 113(2): 293-301, Rosenberg SA et al., Ann Surg 1998; 228(3): 307-19). A particular challenge of rlL2 for cancer treatment is its very short halflife and its side effects.
  • human IL-2 (hlL-2) (optionally as a portion of extended-PK hlL-2) may be naturally occurring h IL-2 or a fragment or variant thereof.
  • h IL-2 comprises the following amino acid sequence, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the following amino acid sequence, or a functional fragment of the following amino acid sequence, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the following amino acid sequence.
  • hlL-2 or a h IL-2 fragment or variant binds to the IL-2 receptor.
  • hlL-2 comprises the following amino acid sequence, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the following amino acid sequence, or a functional fragment of the following amino acid sequence, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the following amino acid sequence.
  • h IL-2 or a hlL-2 fragment or variant binds to the IL-2 receptor.
  • RNA payload may be administered with one or more delivery vehicles that protect the payload from degradation, maximize delivery to on-target cells and minimize exposure to off- target cells.
  • delivery vehicles may complex or encapsulate the payload and include a range of materials, including polymers, lipids and mixtures thereof. In some embodiments, such delivery vehicles may form particles with the payload.
  • a particle relates to a structured entity formed by molecules or molecule complexes, in particular particle forming compounds.
  • a particle is a nucleic acid containing particle such as a particle comprising DNA, RNA or a mixture thereof.
  • the particle contains an envelope (e.g., one or more layers or lamellas) made of one or more types of amphiphilic substances (e.g., amphiphilic lipids).
  • amphiphilic substance means that the substance possesses both hydrophilic and lipophilic properties.
  • the envelope may also comprise additional substances (e.g., additional lipids) which do not have to be amphiphilic.
  • the particle may be a monolameliar or multilamellar structure, wherein the substances constituting the one or more layers or lamellas comprise one or more types of amphiphilic substances (in particular selected from the group consisting of amphiphilic lipids) optionally in combination with additional substances (e.g., additional lipids) which do not have to be amphiphilic.
  • the term "particle” relates to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure. According to the present disclosure, the term “particle” includes nanoparticles.
  • nanoparticle relates to a nano-sized particle comprising at least one particle forming agent, e.g., at least one cationic or cationically ionizable lipid, wherein all three external dimensions of the particle are in the nanoscale, i.e., at least about 1 nm and below about 1000 nm.
  • particle forming agent e.g., at least one cationic or cationically ionizable lipid
  • the size of a particle is its diameter.
  • the particles described herein have a size (such as a diameter) in the range of about 10 to about 2000 nm, such as at least about 15 nm (e.g., at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, or at least about 100 nm) and/or at most about 1900 nm (e.g., at most about 1800 nm, at most about 1700 nm, at most about 1600 nm, at most about 1500 nm, at most about 1400 nm, at most about 1300 nm, at most about 1900 n
  • the particles described herein have a size (such as a diameter) in the range of from about 40 nm to about 200 nm, such as from about 50 nm to about 180 nm, from about 60 nm to about 160 nm, from about 80 nm to about 150 nm or from about 80 nm to about 120 nm.
  • the particles described herein have an average diameter that in some embodiments ranges from about 50 nm to about 1000 nm, from about 50 nm to about 800 nm, from about 50 nm to about 700 nm, from about 50 nm to about 600 nm, from about 50 nm to about 500 nm, from about 50 nm to about 450 nm, from about 50 nm to about 400 nm, from about 50 nm to about 350 nm, from about 50 nm to about 300 nm, from about 50 nm to about 250 nm, from about 50 nm to about 200 nm, from about 100 nm to about 1000 nm, from about 100 nm to about 800 nm, from about 100 nm to about 700 nm, from about 100 nm to about 600 nm, from about 100 nm to about 500 nm, from about 100 nm to about 450 nm, from about 100 nm to about 400 nm, from about
  • the particles described herein have an average diameter that in some embodiments ranges from about 40 nm to about 200 nm, such as from about 50 nm to about 180 nm, from about 60 nm to about 160 nm, from about 80 nm to about 150 nm or from about 80 nm to about 120 nm.
  • Particles described herein may exhibit a polydispersity index (PDI) less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1, or less than about 0.05.
  • the particles can exhibit a polydispersity index in a range of about 0.01 to about 0.4 or about 0.1 to about 0.3.
  • a "nucleic acid particle" can be used to deliver nucleic acid to a target site of interest (e.g., cell, tissue, organ, and the like).
  • a nucleic acid particle may be formed from at least one cationic or cat ionica lly ionizable compound such as a polymer or lipid complexing the nucleic acid. Without intending to be bound by any theory, it is believed that the cationic or cationically ionizable compound combines together with the nucleic acid to form aggregates, and this aggregation results in colloidally stable particles.
  • nucleic acid may be noncovalently associated with a particle.
  • the nucleic acid may be adhered to the outer surface of the particle (surface nucleic acid) and/or may be contained in the particle (encapsulated nucleic acid).
  • the N/P ratio gives the ratio of the nitrogen groups in the lipid to the number of phosphate groups in the nucleic acid. It is correlated to the charge ratio, as the nitrogen atoms (depending on the pH) are usually positively charged and the phosphate groups are negatively charged.
  • the N/P ratio where a charge equilibrium exists, depends on the pH. Lipid formulations are frequently formed at N/P ratios larger than four up to twelve, because positively charged nanoparticles are considered favorable for transfection. In that case, nucleic acid is considered to be completely bound to nanoparticles.
  • Particles described herein can be prepared using a wide range of methods.
  • methods for preparing nucleic acid particles may involve obtaining a colloid from at least one cationic or cationically ionizable lipid and mixing the colloid with nucleic acid to obtain nucleic acid particles.
  • the term "colloid” as used herein relates to a type of homogeneous mixture in which dispersed particles do not settle out.
  • the insoluble particles in the mixture are microscopic, with particle sizes between 1 and 1000 nanometers.
  • the mixture may be termed a colloid or a colloidal suspension. Sometimes the term “colloid” only refers to the particles in the mixture and not the entire suspension.
  • colloids comprising at least one cationic or cationically ionizable lipid methods are applicable herein that are conventionally used for preparing liposomal vesicles and are appropriately adapted.
  • the most commonly used methods for preparing liposomal vesicles share the following fundamental stages: (i) lipids dissolution in organic solvents, (ii) drying of the resultant solution, and (iii) hydration of dried lipid (using various aqueous media).
  • film hydration method lipids are firstly dissolved in a suitable organic solvent, and dried down to yield a thin film at the bottom of the flask. The obtained lipid film is hydrated using an appropriate aqueous medium to produce a liposomal dispersion.
  • an additional downsizing step may be included.
  • Reverse phase evaporation is an alternative method to the film hydration for preparing liposomal vesicles that involves formation of a water-in-oil emulsion between an aqueous phase and an organic phase containing lipids. A brief sonication of this mixture is required for system homogenization. The removal of the organic phase under reduced pressure yields a milky gel that turns subsequently into a liposomal suspension.
  • ethanol injection technique refers to a process, in which an ethanol solution comprising lipids is rapidly injected into an aqueous solution through a needle. This action disperses the lipids throughout the solution and promotes lipid structure formation, for example lipid vesicle formation such as liposome formation.
  • lipoplex particles are obtainable by adding nucleic acid to a colloidal liposome dispersion.
  • colloidal liposome dispersion is, in some embodiments, formed as follows: an ethanol solution comprising lipids, such as cationic or cationically ionizable lipids and additional lipids, is injected into an aqueous solution under stirring.
  • particle forming components or “particle forming agents” relates to any components which form particles, e.g., by associating with a payload.
  • Delivery vehicles such as particle forming agents useful herein include polymers, polymer derivatives, lipids, e.g., as described herein, and mixtures thereof.
  • Such components include any component which can be part of nucleic acid particles, e.g., cationic or cationically ionizable lipids.
  • polymers are commonly used materials for nanoparticle-based delivery.
  • cationic polymers are used to electrostatically condense negatively charged nucleic acid into nanoparticles. These positively charged groups often consist of amines that change their state of protonation in the pH range between 5.5 and 7.5, thought to lead to an ion imbalance that results in endosomal rupture.
  • Polymers such as poly-L-lysine, polyamidoamine, protamine and polyethyleneimine, as well as naturally occurring polymers such as chitosan have all been applied to nucleic acid delivery and are suitable as cationic polymers herein.
  • some investigators have synthesized polymers specifically for nucleic acid delivery. Poly(P-amino esters), in particular, have gained widespread use in nucleic acid delivery owing to their ease of synthesis and biodegradability.
  • Such synthetic polymers are also suitable as cationic polymers herein.
  • a "polymer,” as used herein, is given its ordinary meaning, i.e., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds.
  • the repeat units can all be identical, or in some cases, there can be more than one type of repeat unit present within the polymer.
  • the polymer is biologically derived, i.e., a biopolymer such as a protein.
  • additional moieties can also be present in the polymer, for example targeting moieties.
  • the polymer is said to be a "copolymer.” It is to be understood that the polymer being employed herein can be a copolymer.
  • the repeat units forming the copolymer can be arranged in any fashion. For example, the repeat units can be arranged in a random order, in an alternating order, or as a "block" copolymer, i.e., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc.
  • Block copolymers can have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks.
  • the polymer is biocompatible.
  • Biocompatible polymers are polymers that typically do not result in significant cell death at moderate concentrations.
  • the biocompatible polymer is biodegradable, i.e., the polymer is able to degrade, chemically and/or biologically, within a physiological environment, such as within the body.
  • polymer may be protamine or polyalkyleneimine.
  • protamine refers to any of various strongly basic proteins of relatively low molecular weight that are rich in arginine and are found associated especially with DNA in place of somatic histones in the sperm cells of various animals (as fish).
  • protamine refers to proteins found in fish sperm that are strongly basic, are soluble in water, are not coagulated by heat, and yield chiefly arginine upon hydrolysis. In purified form, they are used in a long-acting formulation of insulin and to neutralize the anticoagulant effects of heparin.
  • protamine as used herein is meant to comprise any protamine amino acid sequence obtained or derived from natural or biological sources including fragments thereof and multimeric forms of said amino acid sequence or fragment thereof as well as (synthesized) polypeptides which are artificial and specifically designed for specific purposes and cannot be isolated from native or biological sources.
  • the polyalkyleneimine comprises polyethylenimine and/or polypropylenimine, preferably polyethyleneimine.
  • a preferred polyalkyleneimine is polyethyleneimine (PEI).
  • the average molecular weight of PEI is preferably 0.75-10 2 to 10 7 Da, preferably 1000 to 10 5 Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, even more preferably 20000 to 25000 Da.
  • linear polyalkyleneimine such as linear polyethyleneimine (PEI).
  • Cationic polymers contemplated for use herein include any cationic polymers which are able to electrostatically bind nucleic acid.
  • cationic polymers contemplated for use herein include any cationic polymers with which nucleic acid can be associated, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.
  • Particles described herein may also comprise polymers other than cationic polymers, i.e., noncationic polymers and/or anionic polymers. Collectively, anionic and neutral polymers are referred to herein as non-cationic polymers.
  • Particles comprising nucleic acid are also referred to as "polyplexes (PLX)" herein.
  • PLX may comprise a lipid component, e.g., the lipid component of a targeting compound.
  • Such particles containing polymer and lipid, e.g., functionalized lipid are also referred to as lipidated polyplexes (LPLX).
  • LPLX lipidated polyplexes
  • the delivery vehicle comprises a polyamine derivative, e.g., a carboxylated polyamine derivative.
  • Polyamines form polycations in solution, which facilitates the complex formation with polyanions such as nucleic acids.
  • a polyamine derivative which is useful herein as delivery vehicle for polyanions comprises: a polyamine moiety comprising a plurality of amino groups; a plurality of carboxylated substituents comprising a carboxyl group bonded via a hydrophobic linker to amino groups of said polyamine moiety; and a plurality of hydrophobic substituents bonded to amino groups of said polyamine moiety.
  • a polyamine derivative which is useful herein as delivery vehicle for polyanions comprises: a polyamine moiety comprising a plurality of amino groups; a plurality of carboxylated substituents comprising a carboxyl group bonded via a hydrophobic linker to amino groups of said polyamine moiety, wherein each of said carboxylated substituents comprises from 6 to 40 carbon atoms, preferably from 6 to 20 carbon atoms, and more preferably from 8 to 16 carbon atoms, and each of said hydrophobic linker may comprise from 1 to 3 heteroatoms selected from O, N, and S; and a plurality of hydrophobic substituents bonded to amino groups of said polyamine moiety, wherein each of said hydrophobic substituents comprises at least 2 carbon atoms, preferably from 6 to 40 carbon atoms, and may comprise from 1 to 3 heteroatoms selected from O, N, and S provided said hydrophobic substituent has at least 6 carbon atoms.
  • each of said carboxylated substituents of said polyamine derivative comprises any one or more of the following moieties as said hydrophobic linker: alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, arylene, and combinations thereof; and/or each of said hydrophobic substituents of said polyamine derivative comprises any one or more of the following moieties: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, and combinations thereof.
  • a polyamine derivative which is useful herein as delivery vehicle for polyanions is a polyalkylenimine derivative having one or more carboxyalkyl substituents comprising from 6 to 40 carbon atoms, and one or more hydrophobic substituents selected from hydrocarbon substituents having at least 2 carbon atoms, preferably from 6 to 40 carbon atoms, wherein each of said hydrophobic substituents may be or may comprise an alkyl group and/or each of said hydrophobic substituents may be or may comprise an aryl group.
  • the polyalkylenimine is selected from the group consisting of polyethylenimines, polypropylenimines, and polybutylenimines.
  • the polyamine moiety of said polyamine derivative may comprise from 4 to 20000 nitrogen atoms, more preferably from 6 to 10000 nitrogen atoms, e.g., from 6 to 1000 nitrogen atoms, or from 6 to 100 nitrogen atoms per polyamine molecule.
  • the polyamine moiety of said polyamine derivative may be a branched polyamine, preferably a branched polyalkylenimine.
  • a carboxylated substitutent comprises one or two carboxyl groups, preferably one carboxyl group.
  • each carboxylated substitutent comprises from 6 to 40 carbon atoms, preferably from 6 to 20 carbon atoms, and more preferably from 8 to 16 carbon atoms.
  • the hydrophobic linkers of said carboxylated substituents may comprise from 1 to 3, preferably, 1 or 2, heteroatoms selected from O, N, and S.
  • the heteroatoms are selected from O and S.
  • 1 or 2 heteroatoms selected from O, N and S, preferably O and S may be contained in the hydrophobic linker.
  • the carboxylated substituents may be carboxyhydrocarbyl groups, or they may be carboxyheterohydrocarbyl groups comprising from 1 to 3 heteroatoms selected from O, N, and S, preferably selected from O and S.
  • the plurality of carboxylated substituents of a molecule of said polyamine derivative there may be exclusively carboxyhydrocarbyl groups, exclusively carboxyheterohydrocarbyl groups, or there may be carboxyhydrocarbyl groups and carboxyheterohydrocarbyl groups.
  • the plurality of carboxylated substituents are all carboxyhydrocarbyl groups. In some embodiments, the plurality of carboxylated substituents are all carboxyheterohydrocarbyl groups.
  • the hydrocarbyl moieties of said carboxyhydrocarbyl groups may be saturated aliphatic hydrocarbyl moieties, unsaturated aliphatic hydrocarbyl moieties, alicyclic hydrocarbyl moieties, aromatic hydrocarbyl moieties, or moieties comprising two or more moieties from the aforementioned list.
  • carboxyhydrocarbyl groups are carboxyalkyl groups, carboxyalkenyl groups, carboxyalkynyl groups, carboxycycloalkyl groups, carboxycycloalkenyl groups, carboxyalkylcycloalkyl groups, carboxycycloalkylalkyl groups, carboxyalkylcycloalkylalkyl groups, carboxyaryl groups, carboxyalkylaryl groups, carboxyarylalkyl groups, and carboxyalkylarylalkyl groups.
  • such carboxyheterohydrocarbyl moieties comprise one or more functional group selected from -0- , -S-, -N(H)C(O)-, -C(O)O- -OC(O)N(H)-, -C(O)-, -C(O)-N(H)-, -N(H)-C(O)-O-, -O-C(O)-, or -S- S- in the hydrophobic linker.
  • the hydrophobic linkers are or comprise alkylene groups such as linear or branched alkylene groups, or the linkers are or comprise cycloalkylene groups.
  • Alkylene groups may be n-alkylene or isoalkylene groups. Examples of alkylene groups are propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, dodecylene, tetradecylene or hexadecylene groups. Examples of cycloalkylene groups are cyclopentylene, cyclohexylene and cycloheptylene groups.
  • alkylcycloalkyl groups are methylcyclopentylene, ethylcyclopentylene, propylcyclopentylene, butylcyclopentylene, pentylcyclopentylene, hexylcylopentylene, methylcyclohexylene, ethylcyclohexylene, propylcyclohexylene, butylcyclohexylene, pentylcyclohexylene and hexylcylohexylene.
  • One or more of these may be combined in a hydrophobic linker.
  • the carboxylated substituents are or comprise carboxyalkyl or carboxycycloalkyl groups comprising from 6 to 20 carbon atoms.
  • Such carboxylated substituents may be selected from the group consisting of carboxy-n-alkyl groups, branched carboxyalkyl groups or cyclic carboxyalkyl groups and their constitution or conformation isomers.
  • the carboxylalkyl groups are radicals of acids selected from hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, 2-cyclohexylacetic acid, 4- cyclohexylbutyric acid, 6-cyclohexylhexanoic acid, 2-(2', 3' or 4' ethylcyclohexyl)-acetic acid or 4-(2', 3' or 4' ethylcyclohexyl)-butyric acid or 6-(2‘, 3' or 4' ethylcyclohexyl)-hexanoic acid.
  • the hydrophobic linkers are or comprise arylene groups and have from 6 to 20 carbon atoms.
  • Aryl groups forming said arylene groups include aromatic hydrocarbyl groups (carbon-only aryl groups) and aromatic heterohydrocarbyl groups (heteroaryl groups). Examples of the former are phenyl, naphthyl, anthracenyl and phenanthryl.
  • nitrogen-containing heteroaryl groups have a pK value of ⁇ 5 for avoiding additional cationic charges at neutral pH.
  • nitrogen-containing heteroaryl groups examples include indolyl groups pyrazinyl groups, pyridazinyl groups, pyrimidinyl groups, cinnolinyl groups, phthalazinyl groups and purinyl groups.
  • oxygen-containing heterohydrocarbyl groups that form hydroxy groups have a pK>12 for avoiding negative charges at neutral pH.
  • alkylaryl groups are methylphenyl (tolyl), ethylphenyl, 4-isopropylphenyl, and xylyl groups.
  • arylalkyl (aralkyl) groups are benzyl, phenylethyl and trityl groups.
  • alkylarylalkyl groups are methylbenzyl and 4-isopropyl benzyl groups.
  • Carboxyarylalkyl moieties may for example be radicals derived from from o, m or p- methyl benzoic acid, or o-, m- or p-ethyl benzoic acid.
  • Carboxyalkylarylalkyl moieties may for example be o-, m- or p-methyl phenylacetic acid.
  • Carboxyalkenylarylalkyl moieties may for example be or from o-, m- or p-methyl cinnamic acid.
  • carboxylated substituents such as those being or comprising carboxyalkyl groups present on the polyamine derivative may be the same or different. For simplicity, they may be the same.
  • the carboxy group of the carboxylated substituent may be bound to any carbon atom of the hydrophobic linker.
  • the carboxy group is bound to a carbon atom as follows: if z is the number of carbon atoms in the longest carbon chain in the carboxylated substituent (such as the carboxyalkyl group) to the carbon atom that is bound to a polyamine nitrogen atom, the carboxy group is bound to a carbon atom at a position that is more than z/2 atom positions away from the polyamine nitrogen, if the carbon atom bound to the polyamine nitrogen is counted as position 1. If the value of z/2 is not an integer, the above definition leads to the position defined by the next integer > z/2.
  • the carboxy group is bound to the carbon atom of the hydrophobic linker that is most remote (in terms of the number of carbon atoms) from the polyamine nitrogen atom to which the hydrophobic linker (alkylene chain in the case of carboxyalkyl groups) is connected.
  • the carboxy group may be bound to the carbon atom that is farthest away from the polyamine nitrogen within the carboxylated substituent (or carboxyalkyl group), such as to the terminal (omega position) carbon atom of the carboxylated substituents (or carboxyalkyl group) in case of a linear carboxylated substituent.
  • the hydrophobic substituents comprise from 2 to 40 carbon atoms, in some embodiments, from 3 to 40 carbon atoms, in some embodiments from 6 to 40 carbon atoms and in some embodiments from 6 to 20 carbon atoms.
  • the hydrophobic substituents may comprise from 1 to 3, preferably 1 or 2, heteroatoms selected from O, N, and S, provided said hydrophobic substituents comprise 6 or more carbon atoms.
  • the heteroatoms are selected from O and S.
  • the hydrophobic substituents may be hydrocarbyl groups or heterohydrocarbyl groups, the latter comprising from 1 to 3 heteroatoms as mentioned before.
  • the plurality of hydrophobic substituents of a molecule of said polyamine derivative there may be exclusively hydrocarbyl groups, exclusively heterohydrocarbyl groups, or there may be hydrocarbyl groups and heterohydrocarbyl groups. In some embodiments, the plurality of hydrophobic substituents are all hydrocarbyl groups. In some embodiments, the plurality of hydrophobic substituents are all heterohydrocarbyl groups.
  • hydrophobic substituents are hydrocarbyl groups, they may be selected from alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, cycloalkenyl groups, cycloalkylalkyl groups, alkylcycloalkyl groups, alkylcycloalkylalkyl groups, aryl groups, alkylaryl groups, arylalkyl groups, and alkylarylalkyl groups and groups comprising two or more groups from the aforementioned list.
  • the hydrophobic substituent comprises 6 or more carbon atoms, it is possible to replace 1, 2 or 3 of the carbon atoms of said hydrocarbyl groups by oxygen, nitrogen or sulfur, preferably oxygen or sulfur, thereby forming heterohydrocarbyl substituents.
  • Such heterohydrocarbyl substituents may comprise functional groups selected from -O-, -S-, -N(H)C(O)-, -C(O)O-, -OC(O)N(H)-, -C(O)-, -C(O)- N(H)-, -N(H)-C(O)-O-, -O-C(O)-, or -S-S-.
  • the hydrophobic substituents are or comprise alkyl groups such as linear or branched alkyl groups, or cycloalkyl groups.
  • Alkyl groups may be n- alkyl or isoalkyl groups. Examples of alkyl groups are propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tetradecyl or hexadecyl groups.
  • Examples of cycloalkyl groups are cyclopentyl, cyclohexyl and cycloheptyl groups.
  • alkenyl groups are propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tetradecenyl and hexadecenyl groups.
  • alkynyl groups are propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl, tetradecynyl and hexadecynyl groups.
  • cycloalkenyl groups are cyclopentenyl, cyclohexenyl and cycloheptenyl groups.
  • Cycloalkylalkyl groups are groups wherein a cycloalkyl group is linked to an alkylene group corresponding to an alkyl group. Examples are cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl, cyclohexylethyl etc.
  • Alkylcycloalkyl groups are groups wherein an alkyl group is linked to a cycloalkylene group corresponding to a cycloalkyl group.
  • alkylcycloalkyl groups are methylcyclopentyl, ethylcyclopentyl, propylcyclopentyl, butylcyclopentyl, pentylcyclopentyl, hexylcylopentyl, methylcyclohexyl, ethylcyclohexyl, propylcyclohexyl, butylcyclohexyl, pentylcyclohexyl and hexylcylohexyl.
  • Alkylcycloalkylalkyl groups are groups wherein an alkyl group is linked to a cycloalkylalkylene group.
  • the hydrophobic substituent comprises an aryl group and has from 6 to 20, preferably from 7 to 15 carbon atoms.
  • Aryl groups include aromatic hydrocarbyl groups (carbon-only aryl groups) and aromatic heterohydrocarbyl groups (heteroaryl groups). Examples of the former are phenyl, naphthyl and phenanthryl.
  • nitrogen-containing heteroaryl groups have a pK value of ⁇ 5 for avoiding additional cationic charges at neutral pH.
  • nitrogen-containing heteroaryl groups examples include indolyl groups pyrazinyl groups, pyridazinyl groups, pyrimidinyl groups, cinnolinyl groups, phthalazinyl groups and purinyl groups.
  • oxygen-containing heterohydrocarbyl groups that form hydroxy groups have a pK>12 for avoiding negative charges at neutral pH.
  • alkylaryl groups are methylphenyl (tolyl), ethylphenyl, 4-isopropylphenyl, methylindolyl and xylyl groups.
  • arylalkyl (aralkyl) groups are benzyl, phenylethyl, indolylmethyl and trityl groups.
  • alkylarylalkyl groups are methylbenzyl and 4- isopropylbenzyl groups.
  • hydrophobic substituents on a molecule of the polyamine derivative may be the same or may be different. For simplicity, they may be the same.
  • the polyamine derivative has a linear polyethylenimine moiety of from 2 to 500 kDa (in terms of number average molecular weight), the carboxylated substituents have from 10 to 16 carbon atoms and are n-alkylcarboxylic acids and the hydrophobic substituents have from 1 to 12 carbon atoms and are alkyls, preferably n-alkyls, and/or alkylarylalkyls.
  • the polyamine derivative has a branched polyethylenimine moiety of from 0.5 to 200 kDa (in terms of number average molecular weight), the carboxylated substituents have from 10 to 16 carbon atoms and are n-alkylcarboxylic acids and the hydrophobic substituents have from 1 to 12 carbon atoms and are alkyls, preferably n-alkyls, and/or alkylarylalkyls.
  • lipid and "lipid-like material” are broadly defined herein as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also frequently denoted as amphiphiles. Lipids are usually insoluble or poorly soluble in water, but soluble in many organic solvents. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases. One of those phases consists of lipid bilayers, as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment.
  • Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s).
  • the hydrophilic groups may comprise polar and/or charged groups and include carbohydrates, phosphate, carboxylic, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other like groups.
  • hydrophobic refers to any a molecule, moiety or group which is substantially immiscible or insoluble in aqueous solution.
  • hydrophobic group includes hydrocarbons having at least 6 carbon atoms.
  • the monovalent radical of a hydrocarbon is referred to as hydrocarbyl herein.
  • the hydrophobic group can have functional groups (e.g., ether, ester, halide, etc.) and atoms other than carbon and hydrogen as long as the group satisfies the condition of being substantially immiscible or insoluble in aqueous solution.
  • hydrocarbon includes non-cyclic, e.g., linear (straight) or branched, hydrocarbyl groups, such as alkyl, alkenyl, or alkynyl as defined herein. It should be appreciated that one or more of the hydrogen atoms in alkyl, alkenyl, or alkynyl may be substituted with other atoms, e.g., halogen, oxygen or sulfur. Unless stated otherwise, hydrocarbon groups can also include a cyclic (alkyl, alkenyl or alkynyl) group or an aryl group, provided that the overall polarity of the hydrocarbon remains relatively nonpolar.
  • alkyl refers to a saturated linear or branched monovalent hydrocarbon moiety which may have one to thirty, typically one to twenty, often six to eighteen carbon atoms.
  • exemplary nonpolar alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, hexyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, and the like.
  • alkenyl refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon double bond in which the total carbon atoms may be two to thirty, typically six to twenty often six to eighteen.
  • the maximal number of carbon-carbon double bonds in the alkenyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenyl group by 2 and, if the number of carbon atoms in the alkenyl group is uneven, rounding the result of the division down to the next integer.
  • the maximum number of carbon-carbon double bonds is 4.
  • the alkenyl group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds.
  • alkynyl refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon triple bond in which the total carbon atoms may be two to thirty, typically six to twenty, often six to eighteen. Alkynyl groups can optionally have one or more carbon-carbon double bonds. Generally, the maximal number of carbon-carbon triple bonds in the alkynyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkynyl group by 2 and, if the number of carbon atoms in the alkynyl group is uneven, rounding the result of the division down to the next integer.
  • the maximum number of carbon-carbon triple bonds is 4.
  • the alkynyl group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, more preferably 1 or 2 carbon-carbon triple bonds.
  • alkylene refers to a saturated linear or branched divalent hydrocarbon moiety which may have one to thirty, typically two to twenty, often four to twelve carbon atoms.
  • exemplary nonpolar alkylene groups include, but are not limited to, methylene, ethylene, trimethylene, hexamethylene, decamethylene, dodecamethylene, tetradecamethylene, hexadecamethylene, octadecmethylene, and the like.
  • alkenylene refers to a linear or branched divalent hydrocarbon moiety having at least one carbon-carbon double bond in which the total carbon atoms may be two to thirty, typically two to twenty, often four to twelve.
  • the maximal number of carboncarbon double bonds in the alkenylene group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenylene group by 2 and, if the number of carbon atoms in the alkenylene group is uneven, rounding the result of the division down to the next integer.
  • the maximum number of carbon-carbon double bonds is 4.
  • the alkenylene group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds.
  • alkynylene refers to a linear or branched divalent hydrocarbon moiety having at least one carbon-carbon triple bond in which the total carbon atoms may be two to thirty, typically two to twenty, often four to twelve. Alkynyl groups can optionally have one or more carbon carbon double bonds.
  • cycloalkyl represents cyclic non-aromatic versions of “alkyl” and "alkenyl” with preferably 3 to 14 carbon atoms, such as 3 to 12 or 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms (such as 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms.
  • Exemplary cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cylcodecyl, cylcodecenyl, and adamantyl.
  • the cycloalkyl group may consist of one ring (monocyclic), two rings (bicyclic), or more than two rings (polycyclic).
  • aryl refers to a monoradical of an aromatic cyclic hydrocarbon.
  • the aryl group contains 3 to 14 (e.g., 5, 6, 7, 8, 9, or 10, such as 5, 6, or 10) carbon atoms which can be arranged in one ring (e.g., phenyl) or two or more condensed rings (e.g., naphthyl).
  • exemplary aryl groups include cyclopropenylium, cyclopentadienyl, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, and phenanthryl.
  • aryl refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. Aryl does not encompass fullerenes.
  • aromatic as used in the context of hydrocarbons means that the whole molecule has to be aromatic.
  • a monocyclic aryl is hydrogenated (either partially or completely) the resulting hydrogenated cyclic structure is classified as cycloalkyl for the purposes of the present disclosure.
  • a bi- or polycyclic aryl such as naphthyl
  • the resulting hydrogenated bi- or polycyclic structure is classified as cycloalkyl for the purposes of the present disclosure (even if one ring, such as in 1,2-dihydronaphthyl, is still aromatic).
  • amphiphilic refers to a molecule having both a polar portion and a non-polar portion. Often, an amphiphilic compound has a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the nonpolar portion is insoluble in water. In addition, the polar portion may have either a formal positive charge, or a formal negative charge. Alternatively, the polar portion may have both a formal positive and a negative charge, and be a zwitterion or inner salt.
  • the amphiphilic compound can be, but is not limited to, one or a plurality of natural or non-natural lipids and lipid-like compounds.
  • lipid-like material lipid-like compound or “lipid-like molecule” relates to substances, in particular amphiphilic substances, that structurally and/or functionally relate to lipids but may not be considered as lipids in a strict sense.
  • the term includes compounds that are able to form amphiphilic layers as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment and includes surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties.
  • the term includes molecules, which comprise hydrophilic and hydrophobic moieties with different structural organization, which may or may not be similar to that of lipids.
  • lipid-like compounds capable of spontaneous integration into cell membranes include functional lipid constructs such as synthetic function-spacer-lipid constructs (FSL), synthetic function-spacer-sterol constructs (FSS) as well as artificial amphipathic molecules.
  • FSL synthetic function-spacer-lipid constructs
  • FSS synthetic function-spacer-sterol constructs
  • Lipids comprising two long alkyl chains and a polar head group are generally cylindrical. The area occupied by the two alkyl chains is similar to the area occupied by the polar head group.
  • Such lipids have low solubility as monomers and tend to aggregate into planar bilayers that are water insoluble.
  • Traditional surfactant monomers comprising only one linear alkyl chain and a hydrophilic head group are generally cone shaped. The hydrophilic head group tends to occupy more molecular space than the linear alkyl chain.
  • surfactants tend to aggregate into spherical or elliptoid micelles that are water soluble. While lipids also have the same general structure as surfactants - a polar hydrophilic head group and a nonpolar hydrophobic tail - lipids differ from surfactants in the shape of the monomers, in the type of aggregates formed in solution, and in the concentration range required for aggregation. As used herein, the term "lipid” is to be construed to cover both lipids and lipid-like materials unless otherwise indicated herein or clearly contradicted by context.
  • lipids may be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from condensation of ketoacyl subunits), sterol lipids and prenol lipids (derived from condensation of isoprene subunits).
  • lipid is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerides.
  • Lipids also encompass molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as steroids, i.e., sterol-containing metabolites such as cholesterol or a derivative thereof.
  • cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.
  • Fatty acids, or fatty acid residues are a diverse group of molecules made of a hydrocarbon chain that terminates with a carboxylic acid group; this arrangement confers the molecule with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in water.
  • the carbon chain typically between four and 24 carbons long, may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains a double bond, there is the possibility of either a cis or trans geometric isomerism, which significantly affects the molecule's configuration. Cis-double bonds cause the fatty acid chain to bend, an effect that is compounded with more cis double bonds in the chain.
  • Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the best-known being the fatty acid triesters of glycerol, called triglycerides.
  • triacylglycerol is sometimes used synonymously with "triglyceride”.
  • the three hydroxyl groups of glycerol are each esterified, typically by different fatty acids.
  • Additional subclasses of glycerolipids are represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic linkage.
  • the glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to two fatty acid-derived "tails" by ester linkages and to one "head” group by a phosphate ester linkage.
  • Examples of glycerophospholipids usually referred to as phospholipids (though sphingomyelins are also classified as phospholipids) are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer).
  • Sphingolipids are a complex family of compounds that share a common structural feature, a sphingoid base backbone.
  • the major sphingoid base in mammals is commonly referred to as sphingosine.
  • Ceramides N-acyl-sphingoid bases
  • the fatty acids are typically saturated or monounsaturated with chain lengths from 16 to 26 carbon atoms.
  • the major phosphosphingolipids of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and fungi have phytoceramide phosphoinositols and mannose-containing headgroups.
  • glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base. Examples of these are the simple and complex glycosphingolipids such as cerebrosides and gangliosides.
  • Sterol lipids such as cholesterol and its derivatives, or tocopherol and its derivatives, are an important component of membrane lipids, along with the glycerophospholipids and sphingomyelins.
  • Saccharolipids describe compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers.
  • a monosaccharide substitutes for the glycerol backbone present in glycerolipids and glycerophospholipids.
  • the most familiar saccharolipids are the acylated glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gram-negative bacteria.
  • Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty-acyl chains. The minimal lipopolysaccharide required for growth in E.
  • Kdo2-Lipid A a hexa-acylated disaccharide of glucosamine that is glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.
  • Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.
  • lipids and lipid-like materials may be cationic, anionic or neutral.
  • Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH.
  • the particles described herein comprise at least one cationic or cationically ionizable lipid as particle forming agent.
  • Cationic or cationically ionizable lipids contemplated for use herein include any cationic or cationically ionizable lipids (including lipid- like materials) which are able to electrostatically bind nucleic acid.
  • cationic or cationically ionizable lipids contemplated for use herein can be associated with nucleic acid, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.
  • a "cationic lipid” refers to a lipid or lipid-like material having a net positive charge.
  • cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries the positive charge.
  • a cationic lipid has a net positive charge only at certain pH, in particular acidic pH, while it has preferably no net positive charge, preferably has no charge, i.e., it is neutral, at a different, preferably higher pH such as physiological pH. This ionizable behavior is thought to enhance efficacy through helping with endosomal escape and reducing toxicity as compared with particles that remain cationic at physiological pH.
  • a “cationically ionizable lipid” refers to a lipid or lipid-like material which has a net positive charge or is neutral, i.e., which is not permanently cationic. Thus, depending on the pH of the composition in which the cationically ionizable lipid is solved, the cationically ionizable lipid is either positively charged or neutral. For purposes of the present disclosure, cationically ionizable lipids are covered by the term “cationic lipid” unless contradicted by the circumstances.
  • the cationic or cationically ionizable lipid comprises a head group which includes at least one nitrogen atom (N) which is positive charged or capable of being protonated, e.g., under physiological conditions.
  • cationic or cationically ionizable lipids include, but are not limited to N,N- dimethyl-2,3-dioleyloxypropylamine (DODMA), l,2-dioleoyl-3-trimethylammonium propane (DOTAP); l,2-di-0-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N— (N',N'- dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); l,2-dioleoyl-3-dimethylammonium-propane (DODAP); l,2-diacyloxy-3- dimethylammonium propanes; l,2-dialkyloxy-3-dimethylammonium propanes; dioctadecyldimethyl ammonium chloride (DODAC), l,2-distearyloxy
  • Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl- [l,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin- MC3-DMA), N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide
  • the cationic or cationically ionizable lipid is DOTMA. In some embodiments, the cationic or cationically ionizable lipid is DODMA.
  • DOTMA is a cationic lipid with a quaternary amine headgroup.
  • the structure of DOTMA may be represented as follows:
  • DODMA is an ionizable cationic lipid with a tertiary amine headgroup.
  • the structure of DODMA may be represented as follows:
  • the cationic or cationically ionizable lipid may comprise from about 10 mol % to about 95 mol %, from about 20 mol % to about 95 mol %, from about 20 mol % to about 90 mol %, from about 30 mol % to about 90 mol %, from about 40 mol % to about 90 mol %, or from about 40 mol % to about 80 mol % of the total lipid present in the particle.
  • the particles described herein may also comprise lipids (including lipid-like materials) other than cationic or cationically ionizable lipids (also collectively referred to herein as cationic lipids), i.e., non-cationic lipids (including non-cationic or non-cationically ionizable lipids or lipid-like materials).
  • cationic lipids also collectively referred to herein as cationic lipids
  • non-cationic lipids including non-cationic or non-cationically ionizable lipids or lipid-like materials.
  • anionic and neutral lipids or lipid-like materials are referred to herein as non-cationic lipids.
  • Optimizing the formulation of nucleic acid particles by addition of other hydrophobic moieties, such as cholesterol and lipids, in addition to a cationic or cationically ionizable lipid may enhance particle stability and efficacy of nucleic acid delivery.
  • One or more additional lipids may or may not affect the overall charge of the nucleic acid particles.
  • the one or more additional lipids are a non-cationic lipid or lipid-like material.
  • the non-cationic lipid may comprise, e.g., one or more anionic lipids and/or neutral lipids.
  • an "anionic lipid” refers to any lipid that is negatively charged at a selected pH.
  • a "neutral lipid” refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH.
  • the nucleic acid particles described herein comprise a cationic or cationically ionizable lipid and one or more additional lipids.
  • the amount of the cationic or cationically ionizable lipid compared to the amount of the one or more additional lipids may affect important nucleic acid particle characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the nucleic acid. Accordingly, in some embodiments, the molar ratio of the cationic or cationically ionizable lipid to the one or more additional lipids is from about 10:0 to about 1:9, about 4:1 to about 1:2, about 4:1 to about 1:1, about 3:1 to about 1:1, or about 3:1 to about 2:1.
  • the one or more additional lipids comprised in the nucleic acid particles described herein comprise one or more of the following: neutral lipids, steroids, and combinations thereof.
  • the one or more additional lipids comprise a neutral lipid which is a phospholipid.
  • the phospholipid is selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins. Specific phospholipids that can be used include, but are not limited to, phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin.
  • Such phospholipids include in particular diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), l,2-di-0-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), l-ole
  • the neutral lipid is selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC. In some embodiments, the neutral lipid is DOPE.
  • the additional lipid comprises one of the following: (1) a phospholipid, (2) cholesterol or a derivative thereof; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof.
  • cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2’-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.
  • the nucleic acid particles described herein comprise (1) a cationic or cationically ionizable lipid, and a phospholipid such as DSPC or DOPE or (2) a cationic or cationically ionizable lipid and a phospholipid such as DSPC or DOPE and cholesterol.
  • the nucleic acid particles described herein comprise (1) DOTMA and DOPE, (2) DOTMA, DOPE and cholesterol, (3) DODMA and DOPE or (4) DODMA, DOPE and cholesterol.
  • DSPC is a neutral phospholipid. The structure of DSPC may be represented as follows:
  • DOPE is a neutral phospholipid.
  • the structure of DOPE may be represented as follows:
  • the structure of cholesterol may be represented as follows:
  • nucleic acid particles described herein do not include a polymer conjugated lipid such as a pegylated lipid.
  • the additional lipid (e.g., one or more phospholipids and/or cholesterol) may comprise from about 0 mol % to about 90 mol %, from about 0 mol % to about 80 mol %, from about 2 mol % to about 80 mol %, from about 5 mol % to about 80 mol %, from about 5 mol % to about 60 mol %, from about 5 mol % to about 50 mol %, from about 7.5 mol % to about 50 mol %, or from about 10 mol % to about 40 mol % of the total lipid present in the particle.
  • the additional lipid (e.g., one or more phospholipids and/or cholesterol) comprises about 10 mol %, about 15 mol %, or about 20 mol % of the total lipid present in the particle.
  • the additional lipid comprises a mixture of: (i) a phospholipid such as DOPE; and (ii) cholesterol or a derivative thereof.
  • the molar ratio of the phospholipid such as DOPE to the cholesterol or a derivative thereof is from about 9:0 to about 1:10, about 2:1 to about 1:4, about 1:1 to about 1:4, or about 1:1 to about 1:3.
  • the particles described herein may comprise at least one polymer- conjugated lipid.
  • the polymer-conjugated lipid comprises an amphiphilic derivative of a polymer which is part of a targeting compound and/or a polymer- conjugated lipid which is not part of a targeting compound.
  • a polymer-conjugated lipid is typically a molecule comprising a lipid portion and a polymer portion conjugated thereto.
  • the polymer of the polymer-conjugated lipid is a polymer as described herein for the targeting compound.
  • a polymer-conjugated lipid is a PEG-conjugated lipid, also referred to herein as pegylated lipid or PEG-lipid.
  • pegylated lipid refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art.
  • a polymer-conjugated lipid is a polysarcosine-conjugated lipid, also referred to herein as sarcosinylated lipid or pSar- lipid.
  • the term “sarcosinylated lipid” refers to a molecule comprising both a lipid portion and a polysarcosine portion.
  • a polymer-conjugated lipid is designed to sterically stabilize a lipid particle by forming a protective hydrophilic layer that shields the hydrophobic lipid layer.
  • a polymer-conjugated lipid can reduce its association with serum proteins and/or the resulting uptake by the reticuloendothelial system when such lipid particles are administered in vivo.
  • the particles described herein comprise a PEG-conjugated lipid.
  • the PEG-conjugated lipid is a lipid having the structure of the following general formula: or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: each of R 12 and R 13 is each independently a straight or branched, alkyl or alkenyl chain containing from 10 to 30 carbon atoms, wherein the alkyl/alkenyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.
  • each of R 12 and R 13 is independently a straight alkyl chain containing from 10 to 18 carbon atoms, preferably from 12 to 16 carbon atoms.
  • R 12 and R 13 are identical. In some embodiments, each of R 12 and R 13 is a straight alkyl chain containing 12 carbon atoms. In some embodiments, each of R 12 and R 13 is a straight alkyl chain containing 14 carbon atoms. In some embodiments, each of R 12 and R 13 is a straight alkyl chain containing 16 carbon atoms.
  • R 12 and R 13 are different. In some embodiments, one of R 12 and R 13 is a straight alkyl chain containing 12 carbon atoms and the other of R 12 and R 13 is a straight alkyl chain containing 14 carbon atoms.
  • w has a mean value ranging from 40 to 50, such as a mean value of 45.
  • w is within a range such that the PEG portion of the pegylated lipid has an average molecular weight of from about 400 to about 6000 g/mol, such as from about 1000 to about 5000 g/mol, from about 1500 to about 4000 g/mol, or from about 2000 to about 3000 g/mol.
  • each of R 12 and R 13 is a straight alkyl chain containing 14 carbon atoms and w has a mean value of 45.
  • PEG-conjugated lipids include, but are not limited to pegylated diacylglycerol (PEG-DAG) such as l-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2' ,3 '-di(tetradecanoyloxy)propyl-l-O-(G)- methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co-methoxy(polyethoxy)ethyl-N-(2,3- di(tetrade
  • the PEG-conjugated lipid is or comprises 2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide.
  • the pegylated lipid has the following structure:
  • the PEG-conjugated lipid is DMG-PEG 2000, e.g., having the following structure:
  • the PEG-conjugated lipid has the following structure: wherein n has a mean value ranging from 30 to 60, such as about 50.
  • the PEG-conjugated lipid is PEG2000-C-DMA which preferably refers to 3-N- [(w-methoxy polyethylene glycol)2000)carbamoyl]-l,2-dimyristyloxy-propylamine (MPEG-(2 kDa)-C-DMA) or methoxy-polyethylene glycol-2,3-bis(tetradecyloxy)propylcarbamate (2000).
  • nucleic acid particles described herein may comprise one or more PEG- conjugated lipids or pegylated lipids as described in WO 2017/075531 and WO 2018/081480, the entire contents of each of which are incorporated herein by reference for the purposes described herein.
  • the pegylated lipid comprises from about 1 mol % to about 10 mol %, preferably from about 1 mol % to about 5 mol %, more preferably from about 1 mol % to about 2.5 mol % of the total lipid present in the nucleic acid compositions/formulations and nucleic acid particles described herein.
  • the RNA described herein may be present in lipoplex particles.
  • Lipoplexes are electrostatic complexes which are generally formed by mixing preformed cationic lipid liposomes with anionic nucleic acid. Formed lipoplexes possess distinct internal arrangements of molecules that arise due to the transformation from liposomal structure into compact nucleic acid lipoplexes.
  • liposomes are self-closed unilamellar or multilamellar vesicular particles wherein the lamellae comprise lipid bilayers and the encapsulated lumen comprises an aqueous phase.
  • a prerequisite for using liposomes for nanoparticle formation is that the lipids in the mixture as required are able to form lamellar (bilayer) phases in the applied aqueous environment.
  • the nucleic acid lipoplex particles include both a cationic lipid and an additional lipid.
  • the cationic lipid is DOTMA and the additional lipid is DOPE.
  • the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In specific embodiments, the molar ratio may be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2:1.
  • Nucleic acid lipoplex particles described herein have an average diameter that in some embodiments ranges from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about 500 nm, or from about 350 nm to about 400 nm.
  • the nucleic acid lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 675 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm.
  • the nucleic acid lipoplex particles have an average diameter that ranges from about 250 nm to about 700 nm. In some embodiments, the nucleic acid lipoplex particles have an average diameter that ranges from about 300 nm to about 500 nm. In an exemplary embodiment, the nucleic acid lipoplex particles have an average diameter of about
  • Embodiments of Lipid nanoparticles are provided.
  • RNA described herein is present in the form of lipid nanoparticles (LNPs).
  • LNP lipid nanoparticles
  • the LNP may comprise any lipid capable of forming a particle to which nucleic acid molecules are attached, or in which nucleic acid molecules are encapsulated.
  • lipid nanoparticles are obtainable from direct mixing of nucleic acid, e.g., RNA, in an aqueous phase with lipids in a phase comprising an organic solvent, such as ethanol.
  • nucleic acid e.g., RNA
  • lipids or lipid mixtures can be used for particle formation, which do not form lamellar (bilayer) phases in water.
  • LNPs typically comprise four components: cationically ionizable lipid, neutral lipids such as phospholipids, a steroid such as cholesterol, and a polymer-conjugated lipid such as PEG-lipid.
  • LNPs may be prepared by mixing lipids dissolved in ethanol with nucleic acid in an aqueous buffer.
  • the LNP comprises from 40 to 60 mol percent, 40 to 55 mol percent, from 45 to 55 mol percent, or from 45 to 50 mol percent of the cationically ionizable lipid.
  • the neutral lipid is present in a concentration ranging from 5 to 15 mol percent, from 7 to 13 mol percent, or from 9 to 11 mol percent.
  • the steroid is present in a concentration ranging from 30 to 50 mol percent, from 30 to 45 mol percent, from 35 to 45 mol percent or from 35 to 43 mol percent.
  • the LNP comprises from 1 to 10 mol percent, from 1 to 5 mol percent, or from 1 to 2.5 mol percent of the polymer-conjugated lipid.
  • the LNP comprises from 45 to 55 mol percent of a cationically ionizable lipid; from 5 to 15 mol percent of a neutral lipid; from 30 to 45 mol percent of a steroid; from 1 to 5 mol percent of a polymer-conjugated lipid; and the nucleic acid, encapsulated within or associated with the lipid nanoparticle.
  • the mol percent is determined based on total mol of lipid present in the lipid nanoparticle. In some embodiments, the mol percent is determined based on total mol of cationically ionizable lipid, neutral lipid, steroid and polymer-conjugated lipid present in the lipid nanoparticle.
  • the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC.
  • the steroid is cholesterol
  • the polymer conjugated lipid is a pegylated lipid, e.g., a pegylated lipid as described above.
  • the cationically ionizable lipid component of the LNPs has the structure of Formula (III):
  • G 1 and G 2 are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene;
  • G 3 is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;
  • R a is H or C1-C12 alkyl
  • R 1 and R 2 are each independently C6-C24 alkyl or C6-C24 alkenyl
  • R 4 is C1-C12 alkyl
  • R 5 is H or Ci-Ce alkyl; and x is 0, 1 or 2.
  • the lipid has one of the following structures (I HA) or (II IB):
  • A is a 3 to 8-membered cycloalkyl or cycloalkylene ring
  • R 6 is, at each occurrence, independently H, OH or C1-C24 alkyl; n is an integer ranging from 1 to 15.
  • the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).
  • the lipid has one of the following structures (IIIC) or (HID):
  • the lipid has one of the following structures (IHE) or (HIF):
  • the lipid has one of the following structures (IIIG), (III H), (Illi), or (IIIJ):
  • n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4.
  • n is 3, 4, 5 or 6.
  • n is 3.
  • n is 4.
  • n is 5.
  • n is 6.
  • y and z are each independently an integer ranging from 2 to 10.
  • y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.
  • R 6 is H. In other of the foregoing embodiments, R 6 is C1-C24 alkyl. In other embodiments, R 6 is OH.
  • G 3 is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G 3 is linear C1-C24 alkylene or linear C1-C24 alkenylene.
  • R 1 or R 2 is C6-C24 alkenyl.
  • R 1 and R 2 each, independently have the following structure: wherein:
  • R 7a and R 7b are, at each occurrence, independently H or C1-C12 alkyl; and a is an integer from 2 to 12, wherein R 7a , R 7b and a are each selected such that R 1 and R 2 each independently comprise from 6 to 20 carbon atoms.
  • a is an integer ranging from 5 to 9 or from 8 to 12.
  • At least one occurrence of R 7a is H.
  • R 7a is H at each occurrence.
  • at least one occurrence of R 7b is Ci-Cs alkyl.
  • Ci-Cs alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.
  • R 1 or R 2 has one of the following structures:
  • R 4 is methyl or ethyl.
  • the cationic lipid of Formula (III) has one of the structures set forth in the table below.
  • RNA described herein is formulated in an LNP composition
  • an LNP composition comprising a cationically ionizable lipid, e.g., a cation ically ionizable lipid as shown above, a neutral lipid, a steroid, and a polymer conjugated lipid.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid of Formula III, a neutral lipid, a steroid, and a polymer conjugated lipid.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid shown in the above tables, a neutral lipid, a steroid, and a polymer conjugated lipid.
  • RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, a neutral lipid, a steroid, and a polymer conjugated lipid.
  • RNA described herein is formulated in an LNP composition comprising ALC-0366, a neutral lipid, a steroid, and a polymer conjugated lipid. In some embodiments, RNA described herein is formulated in an LNP composition comprising ALC-0315, a neutral lipid, a steroid, and a polymer conjugated lipid.
  • the neutral lipid is DSPC.
  • the steroid is cholesterol.
  • the polymer conjugated lipid is a pegylated lipid, e.g., DMG-PEG 2000, PEG2000-C-DMA, or ALC-0159.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid, e.g., a cationically ionizable lipid as shown above, a neutral lipid, a steroid, and a pegylated lipid.
  • a cationically ionizable lipid e.g., a cationically ionizable lipid as shown above
  • a neutral lipid e.g., a steroid, and a pegylated lipid.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid of Formula III, a neutral lipid, a steroid, and a pegylated lipid.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid shown in the above tables, a neutral lipid, a steroid, and a pegylated lipid.
  • RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, a neutral lipid, a steroid, and a pegylated lipid.
  • RNA described herein is formulated in an LNP composition comprising ALC-0366, a neutral lipid, a steroid, and a pegylated lipid.
  • RNA described herein is formulated in an LNP composition comprising ALC-0315, a neutral lipid, a steroid, and a pegylated lipid.
  • the neutral lipid is DSPC.
  • the steroid is cholesterol.
  • the pegylated lipid is DMG-PEG 2000, PEG2000-C-DMA, or ALC-0159.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid, e.g., a cationically ionizable lipid as shown above, DSPC, cholesterol, and a pegylated lipid.
  • a cationically ionizable lipid e.g., a cationically ionizable lipid as shown above, DSPC, cholesterol, and a pegylated lipid.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid of Formula III, DSPC, cholesterol, and a pegylated lipid.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid shown in the above tables, DSPC, cholesterol, and a pegylated lipid. In some embodiments, RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and a pegylated lipid.
  • RNA described herein is formulated in an LNP composition comprising ALC-0366, DSPC, cholesterol, and a pegylated lipid.
  • RNA described herein is formulated in an LNP composition comprising ALC-0315, DSPC, cholesterol, and a pegylated lipid.
  • the pegylated lipid is DMG-PEG 2000, PEG2000-C-DMA, or ALC-0159.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid, e.g., a cationically ionizable lipid as shown above, DSPC, cholesterol, and DMG-PEG 2000.
  • a cationically ionizable lipid e.g., a cationically ionizable lipid as shown above, DSPC, cholesterol, and DMG-PEG 2000.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid of Formula III, DSPC, cholesterol, and DMG-PEG 2000.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid shown in the above tables, DSPC, cholesterol, and DMG-PEG 2000. In some embodiments, RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and DMG-PEG 2000.
  • RNA described herein is formulated in an LNP composition comprising ALC-0366, DSPC, cholesterol, and DMG-PEG 2000.
  • RNA described herein is formulated in an LNP composition comprising ALC-0315, DSPC, cholesterol, and DMG-PEG 2000.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid, e.g., a cationically ionizable lipid as shown above, DSPC, cholesterol, and PEG2000-C-DMA.
  • a cationically ionizable lipid e.g., a cationically ionizable lipid as shown above, DSPC, cholesterol, and PEG2000-C-DMA.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid of Formula III, DSPC, cholesterol, and PEG2000-C-DMA.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid shown in the above tables, DSPC, cholesterol, and PEG2000-C-DMA. In some embodiments, RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and PEG2000-C-DMA. In some embodiments, RNA described herein is formulated in an LNP composition comprising ALC-0366, DSPC, cholesterol, and PEG2000-C-DMA.
  • RNA described herein is formulated in an LNP composition comprising ALC-0315, DSPC, cholesterol, and PEG2000-C-DMA.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid, e.g., a cationically ionizable lipid as shown above, DSPC, cholesterol, and ALC-0159.
  • a cationically ionizable lipid e.g., a cationically ionizable lipid as shown above, DSPC, cholesterol, and ALC-0159.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid of Formula III, DSPC, cholesterol, and ALC-0159.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid shown in the above tables, DSPC, cholesterol, and ALC-0159.
  • RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and ALC-0159.
  • RNA described herein is formulated in an LNP composition comprising ALC-0366, DSPC, cholesterol, and ALC-0159.
  • RNA described herein is formulated in an LNP composition comprising ALC-0315, DSPC, cholesterol, and ALC-0159.
  • 3D-P-DMA (6Z,16Z)-12-((Z)-dec-4-en-l-yl)docosa-6,16-dien-ll-yl 5-
  • ALC-0366 ((3-hydroxypropyl)azanediyl)bis(nonane-9,l-diyl) bis(2-butyloctanoate)
  • ALC-0315 ((4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2-hexyldecanoate) / 6-[N-6-(2- hexyldecanoyloxy)hexyl-N-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate
  • PEG2000-C-DMA 3-N-[(u)-Methoxy polyethylene glycol)2000) carbamoyl]-l,2-dimyristyloxy- propylamine (MPEG-(2 kDa)-C-DMA or Methoxy-polyethylene glycol-2,3- bis(tetradecyloxy)propylcarbamate (2000)) wherein n has a mean value ranging from 30 to 60, such as about 50.
  • ALC-0159 2-[(polyethylene glycol)-2000]-/V,/V-ditetradecylacetamide / 2-[2-(u>-methoxy
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid comprising the following formula (also designated HY-501 herein):
  • the N/P value is preferably at least about 4. In some embodiments, the N/P value ranges from
  • the N/P value is about 6.
  • an RNA payload is delivered specifically to a target immune cell by targeting a target on target immune cells, e.g., an antigen on target immune cells, also referred to herein as "primary target”.
  • target immune cells e.g., an antigen on target immune cells, also referred to herein as "primary target”.
  • the primary target is a structure such as a protein present on the surface of a target immune cell such as a cell surface antigen including a cell surface receptor.
  • Terms such as “expressed on the cell surface”, “associated with the cell surface” or “cell surface molecule” mean that a molecule such as a receptor or antigen is associated with and located at the plasma membrane of a cell, wherein at least a part of the molecule faces the extracellular space of said cell and is accessible from the outside of said cell, e.g., by a binding molecule such as an antibody located outside the cell.
  • a part is preferably at least 4, preferably at least 8, preferably at least 12, more preferably at least 20 amino acids.
  • the association may be direct or indirect.
  • the association may be by one or more transmembrane domains, one or more lipid anchors, or by the interaction with any other protein, lipid, saccharide, or other structure that can be found on the outer leaflet of the plasma membrane of a cell.
  • a molecule associated with the surface of a cell may be a transmembrane protein having an extracellular portion or may be a protein associated with the surface of a cell by interacting with another protein that is a transmembrane protein.
  • Cell surface or “surface of a cell” is used in accordance with its normal meaning in the art, and thus includes the outside of the cell which is accessible to binding by proteins and other molecules.
  • an antigen is expressed on the surface of cells if it is located at the surface of said cells and is accessible to binding by e.g. antigen-specific antibodies added to the cells.
  • an antigen expressed on the surface of cells is an integral membrane protein having an extracellular portion recognized by a binding molecule such as an antibody.
  • extracellular portion or “exodomain” in the context of the present invention refers to a part of a molecule such as a protein that is facing the extracellular space of a cell and preferably is accessible from the outside of said cell, e.g., by binding molecules such as antibodies located outside the cell.

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Abstract

L'invention concerne des agents et des procédés d'administration ciblée d'ARN tel que l'ARNm codant pour un polypeptide comprenant une cytokine ou un variant fonctionnel de celle-ci à des cellules immunitaires pour l'expression du polypeptide. L'administration d'ARN codant pour une cytokine à des cellules immunitaires peut être utile pour l'immunomodulation de cellules immunitaires, en particulier pour induire la prolifération de cellules immunitaires. Dans certains modes de réalisation, l'invention concerne une particule, et un composé de ciblage comprenant une fraction incorporant dans la particule, par exemple, une fraction hydrophobe, et ayant une fraction de liaison fixée de manière covalente à celle-ci. La particule porte une charge utile d'ARN, c'est-à-dire un ARN codant pour un polypeptide comprenant une cytokine ou un variant fonctionnel de celle-ci. Le ciblage d'une cellule immunitaire peut être obtenu par liaison directe ou indirecte du composé de ciblage à des antigènes de surface cellulaire sur la cellule immunitaire cible d'intérêt.
PCT/EP2023/070312 2023-07-21 2023-07-21 Agents et procédés d'administration ciblée de cytokines à des cellules immunitaires Pending WO2025021277A1 (fr)

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