WO2025093665A1 - Particules, compositions et procédés - Google Patents

Particules, compositions et procédés Download PDF

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WO2025093665A1
WO2025093665A1 PCT/EP2024/080804 EP2024080804W WO2025093665A1 WO 2025093665 A1 WO2025093665 A1 WO 2025093665A1 EP 2024080804 W EP2024080804 W EP 2024080804W WO 2025093665 A1 WO2025093665 A1 WO 2025093665A1
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seq
moiety
cdr1
cdr2
cdr3
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Hans-Ulrich Schmoldt
Tanya KAPOOR
Jan Patrick BOGEN
Joycelyn WÜSTEHUBE-LAUSCH
Meike GANGLUFF
Meiyu GAI
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Biontech SE
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Biontech SE
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • A61K47/54—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 organic compound
    • A61K47/541—Organic ions forming an ion pair complex with the pharmacologically or therapeutically active agent
    • 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
    • A61K47/54—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 organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • 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
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • 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
    • A61K47/6873—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 an immunoglobulin; the antibody being an anti-idiotypic antibody
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®

Definitions

  • the present disclosure relates generally to functionalized lipid particles containing moi eties capable of binding Immunoglobulin D (IgD), methods for producing them, and to pharmaceutical compositions containing them and their uses in medicine.
  • IgD Immunoglobulin D
  • the present invention relates to particles comprising single domain antibodies (sdAb) against IgD.
  • the invention further relates to variable heavy chain domain antibodies (VHH), conjugates, functionalized lipid particles, chimeric antigen receptors, and immune cell engagers comprising the sdAbs.
  • VHH variable heavy chain domain antibodies
  • Modalities targeting IgD may be useful in the delivery of a payload to a B cell and the treatment of disease.
  • Immunoglobulin D is an antibody isotype that is co-expressed with IgM on mature B-cells as a part of the B-cell receptor complex together with CD79a and b.
  • IgD can also be detected in circulation in a soluble form, although at very low concentrations (approximately 50x lower compared to IgM and below 0.25 % of total serum Ig; see Nguyen et al. International Reviews of Immunology; 41(2); 107-122).
  • the function of IgD is not very well understood, with early studies having shown that IgD and IgM are functionally interchangeable.
  • IgD knock-outs do not show impairment in B-cell development; however other studies suggest that IgD may control primary immune response and affinity maturation (Amendt et al 2021; Front. Immunol.; 12). Development of agents capable of specifically binding IgD may be useful in a variety of contexts; for example, where it is desirable to target B cells to direct therapeutic and/or diagnostic entities.
  • IgD Immunoglobulin D
  • agents which are capable to specifically binding IgD and for particles capable of carrying such agents which are capable of targeted delivery of a payload to an IgD-expressing cell, such as a B cell.
  • a functionalized particle comprising:
  • a payload such as a nucleic acid payload
  • a payload such as a nucleic acid payload
  • the functionalized particle comprises:
  • a payload such as a nucleic acid payload
  • P is absent or comprises a polymer
  • L comprises a moiety capable of incorporating the compound into the particle, which is attached to Bl when P is absent or to a first end of the polymer P when present;
  • Bl comprises a moiety capable of binding to B2, the moiety Bl being attached to L when P is absent or to a second end of the polymer P when present;
  • XI and X2 are each independently absent or a linking moiety
  • B2 comprises a moiety capable of binding to Bl
  • X3 is absent or a linking moiety
  • B3 comprises a moiety capable of binding to IgD.
  • the functionalized particles comprise:
  • the compound (c) is preferably a compound of Formula (A’): L-X1-P-X2-B’ (A’) wherein:
  • P is absent or comprises a polymer
  • L comprises a moiety capable of incorporating the compound into the particle, L being attached to B when P is absent or to a first end of the polymer P when present;
  • B’ comprises a moiety capable of binding to IgD, the moiety B’ being attached to L when P is absent or to a second end of the polymer P when present;
  • XI is absent or a first linking moiety
  • X2 is absent or a second linking moiety.
  • B’ is preferably selected from the group consisting of an antibody, an antibody-like molecule, a VHH or a peptide. In one embodiment, B’ is a VHH. In one embodiment, B’ is a peptide.
  • step (b) mixing the particle formed in step (a) with the compound of formula (I), such that the compound of formula (I) interacts with the particle comprising the compound of formula (A).
  • a pharmaceutical composition comprising the functionalized lipid particle according to the invention and a carrier.
  • a kit comprising:
  • a payload such as a nucleic acid
  • an in vitro method comprising contacting a cell with the functionalized lipid particle according to the invention.
  • the functionalized lipid particle according to the invention for use in a method of treating a disease or a diagnostic method.
  • the functionalized lipid particle according to the invention for use in the targeted delivery of a payload (such as a nucleic acid payload) to an IgD positive cell.
  • a payload such as a nucleic acid payload
  • the functionalized lipid particle according to the invention for use in the targeted delivery of a payload (such as a nucleic acid payload) to a B cell.
  • a functionalized particle comprising:
  • a moiety capable of binding to a cell surface antigen wherein the first interacting moiety and the second interacting moiety are capable of binding to each other; wherein one of the first interacting moiety and the second interacting moiety comprises a sdAb capable of binding to a ALFA tag peptide, the sdAb comprising CDRs comprising the following sequences:
  • the present inventors have surprisingly determined that functionalized particles (such as lipid particles) comprising a targeting moiety which binds IgD show advantageous properties when targeting B cells.
  • functionalized-lipid particles comprising a targeting moiety which binds IgD show advantages compared to those with targeting moieties against other B cell surface molecules, for example IgM.
  • functionalized nucleic acid-lipid particles comprising a targeting moiety which binds IgD may have increased transfection efficiency compared with those with targeting moieties against IgM.
  • anti-IgD single domain antibodies sdAbs - in particular VHHs
  • the sdAbs are capable of specifically binding to both human and rhesus macaque (Macaca mulatta) IgD; which is beneficial in terms of pre-clinical animal models; such as pre-clinical toxicological assessment.
  • This may also provide advantages when moving from pre- clinical to clinical studies in indications such as HIV/AIDS.
  • the same cross-reactive IgD binder could be used in both studies.
  • the present sdAbs may also have selective binding for IgD compared to other immunoglobulins.
  • the present sdAbs are capable of binding IgD but do not bind, or are essentially incapable of binding, one or more further immunoglobulins.
  • the present sdAbs may be incapable, or essentially incapable of binding IgA, IgE, IgG and/or IgM.
  • the present sdAbs are internalised by a cell following binding to IgD on the cell surface; which may be advantageous for the delivery of a payload to a target cell, for example.
  • Serum from immunized animals was diluted 1 : 100 - 1 : 100000 in assay buffer, and tested for binding against (B) immobilized human IgD Fc-CP protein or (C) rhesus IgD Fc-CP protein via ELISA.
  • CP connecting peptide.
  • FIG. 2 Binding properties of 27 anti IgD VHH candidates derived from screening and HIT ELISA.
  • BBI biolayer interferometry
  • the kinetic binding behavior of the anti-IgD candidates was determined against immobilized IgD Fc parts of human or rhesus origin, either containing or missing the connecting peptide region (CP). Immobilization was done via the terminal His-tag on the IgD Fc domains until a response of 0.7 nm was achieved.
  • VHH-GFP fusions were associated for 600 sec, followed by 600 sec of dissociation. A serial dilution of VHHs was utilized, starting at 50 or 200 nM.
  • Kd, Ka (association rate) and Kdis (dissociation rate) values are given for each candidate.
  • Cell binding and specificity of the IgD VHH was analyzed by incubating 500 and 1000 nM IgD VHH clones with isolated B cells for 30 mins a 4°C. Following the incubation, the cells were washed and stained with the appropriate antibodies. The cells were then fixed and analyzed via FACs. Nb: no binding.
  • FIG. 3 Specificity analysis of selected IgD-Fc binders in VHH-GFP format.
  • Microtiter plates were coated with 5 pmol of either human or rhesus IgD-Fc-CP protein, human IgA, human IgE, human IgG or human IgM. Afterwards, wells were incubated with 1000, 100 and 10 nM of VHHs in 3xFLAG-GFP fusion either diluted in assay buffer only or pre-blocked with 50 % human serum.
  • An anti IgD VHH specific for human IgD only served as a positive control. As a negative control, no VHH was added. Binding towards the different coated antigens was detected using an appropriate secondary antibody.
  • FIG. 4 Overview on the biophysical properties and binding kinetics of 14 selected IgD binders in NbALFA fusion format. Purity was analyzed via SEC and the values indicate the percentage area under the curve of the main peak. Monodispersity (cumulant radius in nm and PDI) was analyzed using DLS with a Prometheus Panta device. The stability was measured via Nano-DSF on a Prometheus Panta device. The binding kinetics of the anti-IgD candidates towards human and rhesus IgD-Fc proteins was determined via BLI. For this, the NbALFA fusions were immobilized onto SAX2.0 sensor tips via biotinylated ALFA-tag peptide for capturing.
  • association was measured over 600 sec using seven different concentrations of the respective IgD-Fc protein of interest starting at 50 nM in a 1 : 1 serial dilution series down to 0.78125 nM.
  • KB buffer without the IgD- Fc was measured. The dissociation was acquired over 600 sec in KB buffer.
  • FIG. 5 Cell binding and internalization: Cells incubated with the various VHH clones at 4°C (for surface binding) and 37°C (for internalization). Analysed via FACs, VHH clones were detected via the FLAG tag.
  • Figure 6 PBMCs treated with algD LNPs PBMCs were treated with 500ng of algD LNPs encapsulating Thy 1.1 RNA.
  • A Example of the FACs analysis of Thy 1.1 expression in the different cell types identified by using CD3 to identify T cells, CD 19 for B cells and CD14 for monocytes.
  • FIGB Graphical representation of the Thy 1.1 expression across the different clones and cell types.
  • FIG. 7 Isolated B cells and PBMCs treated with lOOOng of algD LNPs encapsulating Thy 1.1 RNA and luciferase in the presence of 50% human serum.
  • Isolated B cells treated with the various algD LNPs were analyzed via FACS for the expression of the Thy 1.1 RNA. The plots are gated on CD20&Thyl. l double positive cells following treatment in the presence or absence of human serum.
  • B PBMCs treated with the various algD LNPs were analyzed via FACS for the expression of the Thy 1.1 RNA.
  • T , B and Monocytes are identified by the expression of CD3, CD19 and CD14 respectively.
  • Transfection is detected via expression of Thy 1.1 in each subset.
  • D PBMCs treated with control non-functionalized LNPs and algM LNPs in the presence or absence of human serum were analyzed via FACS for the expression of Thy 1.1 RNA. T-cells, B-cells and Monocytes were identified by the expression of CD3, CD19 and CD14 respectively. Transfection is detected via expression of Thy 1.1 in each subset.
  • FIG. 11 Isolated human B cells were treated with control non-functionalized LNPs and the various algD LNPs for 30 mins in the presence of 50% human serum. Following the particle treatment, cells were incubated overnight and analysed for their activation status via the expression of the activation marker CD86.
  • DLS dynamic light scattering
  • PDI poly dispersity index
  • FIG. 13 - LNPs physicochemical characterization Agarose gel electrophoresis (AGE) showed there was no detectable free cargo Thy 1.1 and Luc mRNA for any of the formulations.
  • the final cargo concentration was 0.1 mg/mL.
  • Two free cargo mixtures (Thy 1.1/Luc mRNA at wt% 1 : 1) with amounts 0.1 pg and 0.5 pg were used as the control samples.
  • Figure 14 - Physicochemical characterization of LNPs A) Illustration of the algD VHH-NbALFA ligand post-functionalization process. NbALFA fusion proteins were immobilized onto ALFA LNP surface via high affinity interaction of NbALFA:ALFA; B) Agarose gel assay for 3 control LNPs (well No. 1-3: 1) without ALFA lipid, 2) with 0.2 mol% ALFA lipid; 3) algM LNP); LNPs functionalized with the top 6 selected algD variants (pNT3129, pNT3133, pNT3193, pNT3194, pNT3197, pNT3198) (well No.
  • FIG. 15 Isolated B cells stained with the proliferation dye CTV were treated with the ALFA (non-functionalized) or the functionalized algD pNT3194 or pNT3198 LNPs. The cells were incubated for 4 days and then analyzed via FACs for their activation status and proliferation via loss in CTV dye.
  • FIG 16 - (A) Freshly prepared human PBMCs (B) Cynomologous Monkey and (C) Rhesus Monkey PBMCs were incubated with 1000 nM of the algD VHH at 4°C for 30 mins, followed by 2X PBS washed. The cells were then stained with CD20 antibodies to distinguish B cell population and with the commercial human and monkey algD antibodies. The VHH were stained with a secondary antibody targeting the nbALFA. The cells were then analysed via FACs for the binding of the VHHs and the commercially available antibodies.
  • FIG 17 Comparison of algD VHHs with commercially available antibodies.
  • Kinetic binding curves of two different exemplary VHH-NbALFA fusions A and B
  • C commercially available anti-monkey IgD antibody
  • D BioLegend #348202
  • Binding was measured via biolayer interferometry on a Sartorius Octet HTX device.
  • Association and dissociation curves are depicted as a solid line, with the fitting results shown as a dotted line. They are separated by a dotted vertical line. The concentration applied during each association step is shown on the right side of each sensorgram with significant responses. No binding could be observed for ABclonal antibody with both antigens and for the BioLegend antibody with rhesus IgD-Fc antigen.
  • FIG. 18 DNA delivery to primary human B cells.
  • B cells treated with LNP formulations encapsulating the sleeping beauty transposon and a reporter gene Venus were analyzed via FACs over a period of 10 days to confirm delivery and insertion of the VENUS DNA.
  • 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 ⁇ 5%, such as ⁇ 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%.
  • the term “about” may in preferred instances indicate deviation from the indicated numerical value by up to 0.3.
  • the expression "substantially free of X”, as used herein, means that the composition described herein is free of X in such manner as it is practically and realistically feasible.
  • the amount of X in the mixture may be less than 1% by weight (e.g., less than 0.5% by weight, less than 0.4% by weight, less than 0.3% by weight, less than 0.2% by weight, less than 0.1% by weight, less than 0.09% by weight, less than 0.08% by weight, less than 0.07% by weight, less than 0.06% by weight, less than 0.05% by weight, less than 0.04% by weight, less than 0.03% by weight, less than 0.02% by weight, less than 0.01% by weight, less than 0.005% by weight, or less than 0.001% by weight), based on the total weight of the mixture. Specific meanings of the term “substantially free” in relation to certain components of the composition are defined herein.
  • physiological pH refers to a pH of about 7.5 or 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.
  • physiological conditions refer to the conditions (in particular pH and temperature) in a living subject, in particular a human. Preferably, physiological conditions mean a physiological pH and/or a temperature of about 37°C.
  • 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 lipid mixture is defined as the ratio of the number of moles of that particular lipid component to the total number of moles of all lipids in the lipid mixture, multiplied by 100.
  • total lipid and/or “total lipid mixture” includes lipids and lipid-like material.
  • hydrocarbyl as used herein relates to a monovalent organic group obtained by removing one H atom from a hydrocarbon molecule.
  • hydrocarbyl groups are non-cyclic, e.g., linear (straight) or branched.
  • Typical examples of hydrocarbyl groups include alkyl, alkenyl, alkynyl, cycloalkyl, aryl groups, and combinations thereof (such as arylalkyl (aralkyl), etc.).
  • hydrocarbyl groups are Ci-40 alkyl (such as Ce-40 alkyl, Ce-30 alkyl, C6-20 alkyl, or C10-20 alkyl), C2-40 alkenyl (such as Ce-40 alkenyl, Ce-30 alkenyl, or C6-20 alkenyl) having 1, 2, or 3 double bonds, aryl, and aryl(Ci-6 alkyl).
  • the hydrocarbyl group is optionally substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.
  • heterohydrocarbyl means a hydrocarbyl group as defined above in which from 1, 2, 3, or 4 carbon atoms in the hydrocarbyl group are replaced by heteroatoms of oxygen, nitrogen, silicon, selenium, phosphorus, or sulfur, preferably O, S, or N.
  • the heterohydrocarbyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.
  • aliphatic refers to a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “cycloaliphatic”), that has a single point or more than one points of attachment to the rest of the molecule.
  • aliphatic groups contain 1-12 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms (e.g., Ci-6).
  • aliphatic groups contain 1-5 aliphatic carbon atoms (e.g., C1-5). In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms (e.g., C1-4). In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms (e.g., C1-3), and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms (e.g., C1-2). Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, or alkynyl groups and hybrids thereof. A preferred aliphatic group is C1-6 alkyl.
  • alkyl refers to a monoradical of a saturated straight or branched hydrocarbon.
  • the alkyl group comprises from 1 to 40, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, carbon atoms, such as 1 to 30, such as 1 to 20 carbon atoms, such as 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, such as 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms.
  • alkyl groups include methyl, ethyl, propyl, iso-propyl (also called 2-propyl or 1 methylethyl), butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, sec-pentyl, neo-pentyl, 1,2- dimethylpropyl, iso-amyl, n-hexyl, iso-hexyl, sec-hexyl, n-heptyl, iso-heptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-undecyl, n-dodecyl, n-dodecyl, n- tridecyl, n-tetradecyl, n-pent
  • a “substituted alkyl” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).
  • the alkyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A. Examples of a substituted alkyl include chloromethyl, dichloromethyl, fluorom ethyl, and difluoromethyl.
  • alkylene refers to a diradical of a saturated straight or branched hydrocarbon.
  • the alkylene group comprises from 1 to 40, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, carbon atoms, such as 1 to 30, such as 1 to 20 carbon atoms, such as 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, such as 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms.
  • Exemplary alkylene groups include methylene, ethylene (i.e., 1,1 -ethylene, 1,2-ethylene), propylene i.e., 1,1- propylene, 1,2-propylene (-CH(CH3)CH2-), 2,2-propylene (-C(CH3)2-), and 1,3- propylene), the butylene isomers (e.g., 1,1-butylene, 1,2-butylene, 2,2-butylene, 1,3- butylene, 2,3-butylene (cis or trans or a mixture thereof), 1,4-butylene, 1 , 1 -isobutylene, 1,2-iso-butylene, and 1,3 -iso-butylene), the pentylene isomers (e.g., 1,1- pentylene, 1,2-pentylene, 1,3-pentylene, 1,4-pentylene, 1,5-pentylene, 1,1-iso- pentylene, 1,1 -sec-pentyl, 1,1-n
  • the straight alkylene moieties having at least 3 carbon atoms and a free valence at each end can also be designated as a multiple of methylene (e.g., 1,4-butylene can also be called tetramethylene).
  • 1,4-butylene can also be called tetramethylene
  • tetramethylene a polymer of polystyrene
  • a “substituted alkylene” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituent may be the same or different).
  • the alkylene is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.
  • alkenyl refers to a monoradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond.
  • 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), z.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds.
  • the alkenyl group comprises from 2 to 40 carbon atoms, such as 2 to 30 carbon atoms, such as 2 to 20 carbon atoms, such as 2 to 12 carbon atoms, such as 2 to 10 carbon atoms, such as 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms.
  • the alkenyl group comprises from 2 to 40, such as 2 to 30, such as 2 to 20, such as 2 to 12, such as 2 to 10 carbon atoms and 1, 2, 3, 4, 5, or 6 (e.g., 1, 2, 3, 4, or 5) carboncarbon double bonds, such as comprises 2 to 8 carbon atoms and 1, 2, 3, or 4 carboncarbon double bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds.
  • the carboncarbon double bond(s) may be in cis (Z) or trans (E) configuration.
  • alkenyl groups include vinyl, 1 -propenyl, 2-propenyl (z.e., allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1 -pentenyl, 2-pentenyl, 3 -pentenyl, 4-pentenyl, 1 -hexenyl, 2-hexenyl, 3- hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5- heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7- nony
  • a “substituted alkenyl” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkenyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkenyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).
  • the alkenyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.
  • alkenylene refers to a diradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond.
  • the maximal number of carbon-carbon 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), z.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds.
  • the alkenylene group comprises from 2 to 12 (such as 2 to 10) carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms.
  • the alkenylene group comprises from 2 to 12 (such as 2 to 10 carbon) atoms and 1, 2, 3, 4, 5, or 6 (such as 1, 2, 3, 4, or 5) carbon-carbon double bonds, more preferably 5 it comprises 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds.
  • the carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration.
  • alkenylene groups include ethen-l,2-diyl, vinylidene (also called ethenylidene), 1- propen-l,2-diyl, 1 -propen- 1,3 -diyl, l-propen-2,3-diyl, allylidene, l-buten-l,2-diyl, 1- buten- 1,3 -diyl, l-buten-l,4-diyl, l-buten-2,3-diyl, l-buten-2,4-diyl, l-buten-3,4-diyl, 2-buten-l,2-diyl, 2-buten- 1,3 -diyl, 2-buten-l,4-diyl, 2-buten-2,3-diyl, 2-buten-2,4- diyl, 2-buten-3,4-diyl, and the like.
  • a “substituted alkenylene” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkenylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 15 up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkenylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different).
  • the alkenylene is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.
  • 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 six to forty, such as six to thirty, typically six to twenty, such as six to eighteen.
  • Alkynyl groups can optionally have one or more carbon-carbon triple bonds.
  • 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.
  • a “substituted alkynyl” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkynyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkynyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).
  • the alkynyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.
  • alkynylene refers to a diradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond.
  • the alkynylene group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon triple bonds.
  • the alkynylene group comprises from 2 to 12 (such as 2 to 10) carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (such as 2, 3, 4, 5, 6, 7, 8,
  • the alkynylene group comprises from 2 to 12 (such as 2 to 10 carbon) atoms and 1, 2, 3, 4, 5, or 6 (such as
  • carbon-carbon triple bonds more preferably it comprises 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon triple bonds, such as 2 to 6 carbon atoms and 1,
  • alkenylene groups include ethyn-l,2-diyl, l-propyn-l,2-diyl, 1-propyn- 1,3 -diyl.
  • the alkenylene is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.
  • cycloalkyl and “cycloalkenyl” represents cyclic non-aromatic versions of “alkyl” and “alkenyl” with preferably 3 to 40, such as 3 to 30, such as 3 to 20, such as 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,
  • cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, and adamantyl.
  • Exemplary cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, cyclononenyl, and cyclodecenyl.
  • the cycloalkyl or cycloalkenyl group may consist of one ring (monocyclic), two rings (bicyclic), or more than two rings (polycyclic).
  • a “substituted cycloalkyl” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a cycloalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the cycloalkyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).
  • the cycloalkyl or cycloalkenyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.
  • cycloalkylene and “cycloalkenylene” represents cyclic non-aromatic versions of “alkylene” and “alkenylene” with preferably 3 to 40, such as 3 to 30, such as 3 to 20, such as 3 to 14 carbon atoms, such as 3 to 12 or 3 to 10 carbon atoms, i.e.,
  • Exemplary cycloalkylene groups include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and cycloheptylene.
  • Exemplary cycloalkylenene groups include cyclopentenylene and cy cl ohexeny 1 ene .
  • 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.
  • Aryl does not encompass fullerenes.
  • a "substituted aryl” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an aryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 5 or up to 10, such as between 1 to 5, 1 to
  • hydrogen atoms of the aryl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).
  • the aryl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.
  • Examples of a substituted aryl include biphenyl, 2-fluorophenyl, 2-chloro-6- methylphenyl, anilinyl, 4-hydroxyphenyl, and methoxyphenyl (z.e., 2-, 3-, or 4- methoxyphenyl).
  • heteroaliphatic or “heteroaliphatic group”, as used herein, denotes an optionally substituted hydrocarbon moiety having, in addition to carbon atoms, from one to five heteroatoms, that may be straight-chain (i.e., unbranched), branched, or cyclic (“heterocyclic”) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic.
  • heteroatom refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen.
  • nitrogen also includes a substituted nitrogen.
  • heteroaliphatic groups contain 1-10 carbon atoms wherein 1-3 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, heteroaliphatic groups contain 1-4 carbon atoms, wherein 1-2 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In yet other embodiments, heteroaliphatic groups contain 1-3 carbon atoms, wherein 1 carbon atom is optionally and independently replaced with a heteroatom selected from oxygen, nitrogen, and sulfur. Suitable heteroaliphatic groups include, but are not limited to, linear or branched, heteroalkyl, heteroalkenyl, and heteroalkynyl groups. For example, a 1- to 10 atom heteroaliphatic group includes the following exemplary groups: -O-CH3, -CH2-O-CH3, -O-CH2-CH2-O- CH2-CH2-O-CH3, and the like.
  • heteroaryl or “heteroaromatic ring” means an aryl group as defined above in which one or more carbon atoms in the aryl group are replaced by heteroatoms of O, S, or N.
  • heteroaryl refers to a five or six-membered aromatic monocyclic ring wherein 1, 2, or 3 carbon atoms are replaced by the same or different heteroatoms of O, N, or S.
  • it means an aromatic bicyclic or tricyclic ring system wherein 1, 2, 3, 4, or 5 carbon atoms are replaced with the same or different heteroatoms of O, N, or S.
  • heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, benzofuranyl, indolyl, isoindolyl, benzothienyl, IH-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotri azolyl
  • Exemplary 5- or 6-memered heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl (e.g., 2-imidazolyl), pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl (e.g., 4-pyridyl), pyrimidinyl, pyrazinyl, triazinyl, and pyridazinyl.
  • a “substituted heteroaryl” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heteroaryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heteroaryl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).
  • the heteroaryl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.
  • heterocyclyl or “heterocyclic ring” means a cycloalkyl group as defined above in which from 1, 2, 3, or 4 carbon atoms in the cycloalkyl group are replaced by heteroatoms of oxygen, nitrogen, silicon, selenium, phosphorus, or sulfur, preferably O, S, or N.
  • a heterocyclyl group has preferably 1 or 2 rings containing from 3 to 10, such as 3, 4, 5, 6, or 7, ring atoms.
  • the maximum number of O atoms is 1, the 5 maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2.
  • alkylcycloalkyl means a cycloalkyl group, as defined above, which is substituted with an alkyl group, as defined above, the cycloalkyl portion being connected to the rest of the molecule.
  • alkylcycloalkyl means a cycloalkyl group, as defined above, which is substituted with an alkyl group, as defined above, the cycloalkyl portion being connected to the rest of the molecule.
  • Each of the cycloalkyl and alkyl portions of the group may take any of the broadest or preferred meanings recited above.
  • a “substituted alkylcycloalkyl” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a alkylcycloalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or cycloalkyl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).
  • the alkylcycloalkyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.
  • a "substituted cycloalkylalkyl” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a cycloalkylalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or cycloalkyl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).
  • the cycloalkylalkyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.
  • alkylcycloalkylalkyl means an alkyl group, as defined above, which is substituted with a cycloalkyl group, as defined above, the alkyl portion being connected to the rest of the molecule and the cycloalkyl portion in turn being substituted with a further alkyl group.
  • cycloalkyl and alkyl portions of the group may take any of the broadest or preferred meanings recited above.
  • a "substituted alkylcycloalkylalkyl” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a alkylcycloalkylalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or cycloalkyl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).
  • the alkylcycloalkylalkyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.
  • a “substituted alkylaryl” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkylaryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or aryl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).
  • the alkylaryl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.
  • arylalkyl means an alkyl group, as defined above, which is substituted with an aryl group, as defined above, the alkyl portion being connected to the rest of the molecule.
  • aryl and alkyl portions of the group may take any of the broadest or preferred meanings recited above.
  • a “substituted arylalkyl” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a arylalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or aryl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).
  • the arylalkyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.
  • alkylheteroaryl means a heteroaryl group, as defined above, which is substituted with an alkyl group, as defined above, the heteroaryl portion being connected to the rest of the molecule.
  • Each of the heteroaryl and alkyl portions of the group may take any of the broadest or preferred meanings recited above.
  • a “substituted alkylheteroaryl” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkylheteroaryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or heteroaryl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).
  • the alkylheteroaryl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.
  • a “substituted heteroarylalkyl” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heteroarylalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or heteroaryl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).
  • the heteroarylalkyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.
  • alkylheterocyclyl means a heterocyclyl group, as defined above, which is substituted with an alkyl group, as defined above, the heteroaryl portion being connected to the rest of the molecule.
  • Each of the heterocyclyl and alkyl portions of the group may take any of the broadest or preferred meanings recited above.
  • a "substituted alkylheterocyclyl” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkylheterocyclyl group, e.g., 1, 2, 3, 4, 5, 6,
  • the alkylheterocyclyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.
  • heterocyclylalkyl means an alkyl group, as defined above, which is substituted with a heterocyclyl group, as defined above, the alkyl portion being connected to the rest of the molecule.
  • heterocyclyl and alkyl portions of the group may take any of the broadest or preferred meanings recited above.
  • a "substituted heterocyclylalkyl” means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heterocyclylalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7,
  • the heterocyclylalkyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.
  • organosulfuric acid or “sulfate” means a compound of formula R-OSO2- OH, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect).
  • R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkyl
  • sulfate is used when the group is deprotonated. Depending on the pH, the sulfate group may be protonated or deprotonated (in the anionic amphiphiles as defined below, the sulfonic acid group is typically deprotonated at physiological pH).
  • sulfonic acid or “sulfonate” means a compound of formula R-SO2-OH, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect).
  • R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcyclo
  • sulfonate is used when the group is deprotonated. Depending on the pH, the sulfonate group may be protonated or deprotonated (in the anionic amphiphiles as defined below, the sulfonate group is typically deprotonated at physiological pH).
  • carboxylic acid or “carboxylate” means a compound of formula R-CO2H, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect).
  • R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl
  • carboxylate is used when the group is deprotonated.
  • the carboxylic acid may be protonated or deprotonated (in the anionic amphiphiles as defined below, the carboxylic acid group is typically protonated at acidic pH and deprotonated at neutral or alkaline pH).
  • hydroxy carboxylic acid or “hydroxy carboxylate” means a compound of formula R-CO2H, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect), which is substituted by one or more (preferably 1 to 5, such as 1, 2 or 3) hydroxy groups.
  • R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl,
  • hydroxy carboxylate is used when the group is deprotonated.
  • the hydroxy carboxylic acid may be protonated or deprotonated (in the anionic amphiphiles as defined below, the carboxylic acid group is typically protonated at acidic pH and deprotonated at neutral or alkaline pH).
  • ester moiety may have the structure R-C(O)O- or R-OC(O)-, where R is as defined above.
  • each of both ends of the ester structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker).
  • the phosphate group may be protonated or deprotonated (in the anionic amphiphiles as defined below, the phosphate group is typically deprotonated at physiological pH).
  • the phosphonate group may be protonated or deprotonated (in the anionic amphiphiles as defined below, the phosphonate group is typically deprotonated at physiological pH).
  • Halo means fluoro (-F), chloro (-C1), bromo (-Br) or iodo (-1).
  • “Amine” means the group -NR2, wherein each R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect), and is preferably an alkyl group, such as a C1-6 alkyl group.
  • both groups R are hydrogen
  • the amine group is a primary amine group.
  • the amine group is a secondary amine group.
  • the amine group is a tertiary amine group.
  • a “quaternary ammonium” salt is a compound containing a group -N + R3, wherein each R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect), and is preferably an alkyl group, such as a C1-6 alkyl group.
  • a quaternary ammonium salt carries a constitutive positive charge (as defined herein) at all
  • “Hydroxyl” - means the group -OH.
  • “Sulfhydryl” - means the group -SH.
  • “Nitro” means the group -NO2.
  • “Ether” means an oxygen atom to which two hydrocarbyl or heterohydrocarbyl groups, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl groups (all as defined above, either in a broadest aspect or a preferred aspect) are attached.
  • the ether may be a cyclic ether, wherein the two hydrocarbyl groups together form a ring
  • Thioether means or a bivalent linkage of formula -S- where both connected moieties are via the sulfur atom, or a group of formula -SR wherein R is a Ci-io alkyl group.
  • “Disulfide” means or a bivalent linkage of formula - S-S- where one moiety is connected to the first sulfur atom and another to the second sulfur atom.
  • guanidinium group is a protonated guanidine
  • an ammonium group is a protonated ammonia or a protonated primary, secondary tertiary amine
  • an imidazolium group is a protonated imidazole
  • a pyridinium group is a protonated pyridine
  • an amidinium group is a protonated amidine
  • a piperazinium group is a protonated piperazine.
  • Carbohydrate means a compound having the empirical formula Cm(H20)n where m may or may not be different from n.
  • the term “carbohydrate residue” or “carbohydrate moiety” defines a residue attached to another atom, where one hydrogen atom of the carbohydrate is replaced by a bond attached to the rest of the molecule.
  • the carbohydrate moiety may be a monosaccharide moiety.
  • the monosaccharide moiety may have the D- or L-configuration.
  • the monosaccharide moiety may be an aldose or ketose moiety.
  • the monosaccharide moiety may have 3 to 8, preferably 4 to 6, more preferably 5 or 6, carbon atoms.
  • the monosaccharide moiety is a hexose moiety (i.e. it has 6 carbon atoms), examples of which include aldohexoses such as glucose, galactose, allose, altrose, mannose, gulose, idose and talose, and ketohexoses such as fructose and sorbose.
  • aldohexoses such as glucose, galactose, allose, altrose, mannose, gulose, idose and talose
  • ketohexoses such as fructose and sorbose.
  • the hexose moiety is a glucose moiety.
  • the monosaccharide moiety is a pentose moiety (i.e. it has 5 carbon atoms), such as ribose, arabinose, xylose or lyxose.
  • the pentose moiety is an arabinose or xylose moiety.
  • the carbohydrate may be a higher saccharide (i.e. a di-, or oligosaccharide) comprising more than one monosaccharide moiety joined together by glycoside bonds.
  • the glycoside bonds may be l-a,l'-a glycoside bonds, l,2'-gly coside bonds (which maybe l-a2’ or 1 '-P-2' glycoside bonds), l,3'-glycoside bonds (which may be l-a-3' or 1-P- 3 '-glycoside bonds), l,4'-gly coside bonds (which may be l-a-4' or l-P-4'-gly coside bonds), l,6'-gly coside bonds (which may be l-a-6' or l-P-6'-gly coside bonds), or any combination thereof.
  • the higher saccharide comprises 2 monosaccharide units (i.e. is a di saccharide).
  • suitable disaccharides include maltose, isomaltose, isomaltulose, lactose, sucrose, cellobiose, nigerose, kojibiose, trehalose and trehalulose.
  • the higher saccharide comprises 3 to 10 monosaccharide units (i.e. is an oligosaccharide) in a chain, which may be branched or unbranched.
  • the oligosaccharide comprises 3 to 8, more preferably 3 to 6, monosaccharide units.
  • Suitable oligosaccharides include maltodextrin, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, melezitose, cellotriose, cellotetraose, cellopentaose, cellohexaose and celloheptaose.
  • “List A” substituents are selected from the group consisting of Ci-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 6- to 14-membered (such as 6- to 10-membered) aryl, 3- to 14- membered (such as 5- or 6- membered) heteroaryl, 3- to 14-membered (such as 3- to 7-membered) cycloalkyl, 3- to 14-membered (such as 3- to 7-membered) heterocyclyl, halogen, -CN, azido, -NO2, -OR’, -N(R’)2, -S(0)o-2R’, -S(O)I-2OR’, -OS(O)I- 2 R’, -OS(O)I- 2 OR’, -S(O)I- 2 N(R’)2, -OS(O)I- 2 N(R’)2, -N R’)S(O)I- 2 R’, -N(R’)
  • the present invention provides in one aspect functionalized particles as described herein.
  • the functionalized particles are capable of delivering a payload (such as a nucleic acid payload) to a target.
  • the particles act to stabilise and encapsulate the payload (such as a nucleic acid) to enable it to be delivered into a cell while facilitating its uptake into the cell and release into the cytosol.
  • the term "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.
  • nanoparticle relates to a nano-sized particle comprising at least one particle forming agent, e.g., at least one cationic or cationically ionizable lipid or a cationic polymer, wherein all three external dimensions of the particle are in the nanoscale, z.e., at least about 1 nm and below about 1000 nm.
  • 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 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.
  • 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.
  • LNPs may be understood as oil-in-water emulsions in which the LNP core materials are preferably in liquid state and hence have a melting point below body temperature.
  • LNPs thus typically comprise a central complex of lipid and optionally a payload, such as a nucleic acid (e.g., RNA (such as mRNA), DNA or mixtures thereof) embedded in a disordered, non-lamellar phase made of lipid.
  • RNA such as mRNA
  • Lipid nanoparticles are obtainable from combining a payload, such as a nucleic acid, with lipids.
  • the lipids used for LNP formation typically do not form lamellar (bilayer) phases in water under physiological conditions.
  • the LNPs typically do not comprise or encapsulate an aqueous core.
  • the LNPs typically comprise a lipidic (or oily) core.
  • the lipid nanoparticles described herein have an average size (such as a diameter) that in some embodiments ranges from 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 1900 n
  • the functionalized particle of the present disclosure is a lipoplex (LPX).
  • the one or more particle forming components comprises a cationic or cationically ionizable lipid.
  • Lipoplexes (LPX) are electrostatic complexes which are generally formed by mixing preformed particles containing cationic or cationically ionizable lipid with an anionic payload (such as a nucleic acid). Formed lipoplexes possess distinct internal arrangements of molecules that arise due to the transformation from liposomal structure into compact nucleic acid lipoplexes.
  • the functionalized particle of the present disclosure is a polyplex (PLX).
  • the one or more particle forming components comprises a cationic polymer, as defined and exemplified below.
  • a cationic polymer is capable of electrostatically condensing a negatively charged payload (such as a nucleic acid) into particles.
  • Polyplexes can also contain anionic polymers, as described below.
  • Polyplexes can also contain neutral (e.g., hydrophilic) polymers, such as stealth polymers (e.g., PEG, pSar, pAEEA, etc.) as described below.
  • the functionalized particle of the present disclosure is a lipidated polyplex (LPLX).
  • the one or more particle forming components comprises a cationic polymer, as defined and exemplified below.
  • the cationic polymer used for LPLX formation comprises a hydrophobic portion to facilitate interaction with the lipid component of the LPLX (e.g., Viromers), as further described below.
  • LPLX can also contain neutral (e.g., hydrophilic) polymers, such as stealth polymers (e.g., PEG, pSar, pAEEA, etc.), as described below.
  • Lipidated polyplexes also contain lipids, as defined and exemplified below.
  • the functionalized particles of the present invention typically comprise:
  • a payload such as a nucleic acid payload
  • the functionalized particles comprise:
  • a payload such as a nucleic acid payload
  • the functionalized particles comprise:
  • P is absent or comprises a polymer
  • L comprises a moiety capable of incorporating the compound into the particle], which is attached to Bl when P is absent or to a first end of the polymer P when present;
  • Bl comprises a moiety capable of binding to B2, the moiety Bl being attached to L when P is absent or to a second end of the polymer P when present;
  • XI and X2 are each independently absent or a linking moiety
  • B2 comprises a moiety capable of binding to Bl
  • X3 is absent or a linking moiety
  • B3 comprises a moiety capable of binding to IgD.
  • the particle as described herein, comprises a compound of formula (A’), wherein the moiety B’ of the compound of formula (A’) is a moiety binding to IgD on target cells.
  • the compound of formula (A’) functionalizes the particles such that, in use, a connection can be made between the moiety B’ of the compound of formula (A’) and IgD, to enable the payload comprised in the loaded-lipid particle to be delivered to a target cell expressing IgD or presenting IgD on its surface.
  • the particle, as described herein, comprising a connector compound, as defined herein is contacted with a docking compound, as defined herein, such that the second interacting moiety of the docking compound of formula (I) interacts with the first interacting moiety of the connector compound.
  • the particle, as described herein, comprising a compound of formula (A) is contacted with a compound of formula (I), as defined herein, such that the moiety B2 of the compound of formula (I) interacts with the moiety Bl of the compound of formula (A).
  • the compound of formula (I) interacts with, or binds to, the loaded lipid particle, as described herein.
  • the terms “interacts with” and “binds to” may be used interchangeably in this context.
  • the docking compound e.g., the compound of formula (I)
  • the interaction between the connector compound and the docking compound functionalizes the particles such that, in use, a connection can be made between the docking compound and IgD, e.g., IgD on target cells, to enable the payload (such as a nucleic acid) comprised in the functionalized particle to be delivered to a target cell expressing IgD or presenting IgD on its surface.
  • the moiety Bl comprises an antibody, an antibody-like molecule, or a VHH, and the moiety B2 is a peptide.
  • the moiety B2 comprises an antibody, an antibody-like molecule, or a VHH, and the moiety Bl is a peptide.
  • the moiety B3 is a single domain antibody (sdAb) capable of binding IgD.
  • the sdAb is a variable heavy chain domain antibody (VHH), a heavy chain variable (VH) domain, or a variable new antigen receptor antibody (VNAR).
  • the sdAb is a VHH.
  • the compound of the formula B2-X3-B3 comprises a peptide or polypeptide.
  • the moiety capable of binding to IgD comprises an antibody or antibody-like molecule.
  • the moiety Bl comprises a peptide tag and the moiety B2 comprises a moiety capable of binding to the peptide tag. In one embodiment, the moiety B2 comprises a peptide tag and the moiety Bl comprises a moiety capable of binding to the peptide tag.
  • the moiety capable of binding to a peptide tag comprises an antibody or antibody-like molecule.
  • the peptide tag comprises an ALFA-tag, as defined below.
  • the moiety Bl of the compound of formula (A) comprises a peptide tag
  • the moiety B2 of the compound of formula (I) binds to the compound of formula (A) via the peptide tag.
  • the peptide tag preferably comprises an ALFA-tag.
  • the moiety B2 of the compound of formula (I) is preferably a VHH capable of binding to the ALFA-tag.
  • the moiety B2 of the compound of formula (I) comprises a peptide tag
  • the moiety Bl of the compound of formula (A) binds to the compound of formula (I) via the peptide tag.
  • the peptide tag preferably comprises an ALFA-tag.
  • the moiety Bl of the compound of formula (A) is a VHH capable of binding to the ALFA-tag.
  • the functionalized lipid particles as defined herein include a payload.
  • a payload comprises a therapeutic or diagnostic moiety.
  • the payload may be RNA, such as mRNA.
  • the payload may be DNA.
  • the payload may be a mixture of RNA, such as mRNA, and DNA.
  • the payload can be a therapeutic agent such as a pharmaceutically active agent.
  • a therapeutic agent such as a pharmaceutically active agent.
  • pharmaceutically active agents are known to the skilled person and provided herein.
  • a therapeutic agent can optionally also comprise a detectable label, as defined below.
  • the agents and methods described herein are used for targeted therapy. This is achieved by making use of a payload comprising one or more pharmaceutically active agents (e.g., a drug or a radioactive isotope for radiation therapy).
  • a payload comprising one or more pharmaceutically active agents (e.g., a drug or a radioactive isotope for radiation therapy).
  • pharmaceutically active agent relates to any agent such as compound or cell being therapeutically effective when administered to an individual.
  • pharmaceutically active agent further relates to any agent that changes, preferably cures, alleviates or partially arrests the clinical manifestations of a given disease and its complications in a therapeutic intervention comprising the administration of said agent.
  • the payload is a nucleic acid, such as pharmaceutically active RNA (e.g. mRNA) or DNA, as defined in more detail below.
  • a pharmaceutically active agent comprises a pharmaceutically active peptide or protein, as defined in more detail below.
  • a payload comprises a nucleic acid, as defined in more detail below.
  • the functionalized particles allow the nucleic acid payload, to be delivered to target cells to genetically modify the target cells and enable the target cells to express a biomolecule, e.g., peptide or protein, encoded by the nucleic acid.
  • the nucleic acid is nucleic acid encoding an antigen receptor.
  • the target cells may be immune cells or immune effector cells.
  • the payload is a gene editing reagent or tool (e.g., transposon (such as sleeping beauty or piggy bac) or CRISPR/Cas (or related) based system).
  • transposase such as sleeping beauty or piggy bac
  • CRISPR/Cas or related
  • Such tools e.g., transposase, gene editing tools like CRISPR/Cas9 for genomic integration/editing may be delivered as protein or coding nucleic acid (DNA or RNA).
  • the payload may comprise one or more nucleic acids (e.g. RNA or mRNA) encoding for a transposase (e.g., a Sleeping Beauty transposase, such as SB100X), and one or more nucleic acid (e.g., DNA) transposon(s).
  • the transposon typically encodes a gene of interest, to be inserted into the genome.
  • the payload may comprise one or more nucleic acids (e.g. RNA or mRNA) encoding for a Cas endonuclease (e.g. Cas9) and a gRNA.
  • the payload may comprise a peptide or protein comprising a Cas endonuclease (e.g., Cas9) function and one or more nucleic acids (e.g. RNA, mRNA, DNA or mixtures thereof) comprising a gRNA.
  • Cas is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence.
  • Cas enzymes, together with CRISPR sequences, form the basis of a technology known as CRISPR-Cas that can be used to edit genes within organisms. This editing process has a wide variety of applications including basic biological research, development of biotechnology products, and treatment of diseases.
  • CRISPR/Cas is a target-specific technique that can introduce gene knock out or knock in depending on the double strand repair pathway.
  • the targeting specificity of CRISPR-Cas is determined by the 20-nt sequence at the 5' end of the guide RNA (gRNA).
  • the desired target sequence must precede the protospacer adjacent motif (PAM) which is a short DNA sequence usually 2-6 base pairs in length that follows the DNA region targeted for cleavage by CRISPR-Cas.
  • the PAM is required for a Cas nuclease to cut and is generally found 3-4 nucleotides downstream from the cut site. After base pairing of the gRNA to the target, Cas mediates a double strand break about 3 -nt upstream of PAM.
  • a payload comprises a compound useful in radiation therapy and/or chemotherapy. In some embodiments, a payload comprises a chemotherapeutic compound.
  • Chemotherapy is a type of cancer treatment that uses one or more anti-cancer drugs (chemotherapeutic agents), usually as part of a standardized chemotherapy regimen.
  • chemotherapy has come to connote non-specific usage of intracellular poisons to inhibit mitosis. The connotation excludes more selective agents that block extracellular signals (signal transduction).
  • therapies with specific molecular or genetic targets, which inhibit growth-promoting signals from classic endocrine hormones (primarily estrogens for breast cancer and androgens for prostate cancer) are now called hormonal therapies.
  • other inhibitions of growthsignals like those associated with receptor tyrosine kinases are referred to as targeted therapy.
  • chemotherapeutic agents are cytotoxic by means of interfering with cell division (mitosis) but cancer cells vary widely in their susceptibility to these agents. To a large extent, chemotherapy can be thought of as a way to damage or stress cells, which may then lead to cell death if apoptosis is initiated.
  • Chemotherapeutic agents include alkylating agents, antimetabolites, anti -microtubule agents, topoisomerase inhibitors, and cytotoxic antibiotics.
  • Alkylating agents have the ability to alkylate many molecules, including proteins, RNA and DNA.
  • the subtypes of alkylating agents are the nitrogen mustards, nitrosoureas, tetrazines, aziridines, cisplatins and derivatives, and non-classical alkylating agents.
  • Nitrogen mustards include mechlorethamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide and busulfan.
  • Nitrosoureas include N-Nitroso-N- methylurea (MNU), carmustine (BCNU), lomustine (CCNU) and semustine (MeCCNU), fotemustine and streptozotocin.
  • Tetrazines include dacarbazine, mitozolomide and temozolomide.
  • Aziridines include thiotepa, mytomycin and diaziquone (AZQ).
  • Cisplatin and derivatives include cisplatin, carboplatin and oxaliplatin. They impair cell function by forming covalent bonds with the amino, carboxyl, sulfhydryl, and phosphate groups in biologically important molecules.
  • Non- classical alkylating agents include procarbazine and hexamethylmelamine. In one particularly preferred embodiment, the alkylating agent is cyclophosphamide.
  • Anti-metabolites are a group of molecules that impede DNA and RNA synthesis. Many of them have a similar structure to the building blocks of DNA and RNA. Antimetabolites resemble either nucleobases or nucleosides, but have altered chemical groups. These drugs exert their effect by either blocking the enzymes required for DNA synthesis or becoming incorporated into DNA or RNA. Subtypes of the antimetabolites are the anti-folates, fluoropyrimidines, deoxynucleoside analogues and thiopurines.
  • the anti-folates include methotrexate and pemetrexed.
  • the fluoropyrimidines include fluorouracil and capecitabine.
  • the deoxynucleoside analogues include cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, and pentostatin.
  • the thiopurines include thioguanine and mercaptopurine.
  • Anti -microtubule agents block cell division by preventing microtubule function.
  • the vinca alkaloids prevent the formation of the microtubules, whereas the taxanes prevent the microtubule disassembly.
  • Vinca alkaloids include vinorelbine, vindesine, and vinflunine.
  • Taxanes include docetaxel (Taxotere) and paclitaxel (Taxol).
  • Topoisomerase inhibitors are drugs that affect the activity of two enzymes: topoisomerase I and topoisomerase II and include irinotecan, topotecan, camptothecin, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone, and aclarubicin.
  • the cytotoxic antibiotics are a varied group of drugs that have various mechanisms of action.
  • the common theme that they share in their chemotherapy indication is that they interrupt cell division.
  • the most important subgroup is the anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin pirarubicin, and aclarubicin) and the bleomycins; other prominent examples include mitomycin C, mitoxantrone, and actinomycin.
  • the payload is a detectable label.
  • a "detectable label” as used herein relates to a compound which allows its detection, e.g., when present in a cell, tissue or organism.
  • One type of detectable label envisaged within the context of the present disclosure is a contrast providing agent. Different types of detectable labels are envisaged within the context of the present disclosure and are described herein below.
  • the agents and methods of the present disclosure are used in imaging, especially medical imaging.
  • an imaging probe comprising one or more detectable labels.
  • detectable labels of the imaging probe are contrast-providing moieties used in traditional imaging systems such as MRI-imageable constructs, spin labels, optical labels, ultrasound- responsive constructs, X-ray-responsive moieties, radionuclides, (bio)luminescent and FRET -type dyes.
  • Exemplary detectable labels envisaged within the context of the present disclosure include, but are not limited to, fluorescent molecules, e.g.
  • autofluorescent molecules molecules that fluoresce upon contact with a reagent, etc., radioactive labels; biotin, e.g., to be detected through binding of biotin by avidin; fluorescent tags, imaging constructs for MRI comprising paramagnetic metal, imaging reagents and the like.
  • the radionuclide used for imaging can be, for example, an isotope selected from the group consisting of 3 H, n C, 13 N, 15 O, 18 F, 19 F, 51 Cr, 52 Fe, 52 Mn, 55 Co, 60 Cu, 61 Cu, 62 Zn, 62 Cu, 63 Zn, 64 Cu, 66 Ga, 67 Ga, 68 Ga, 70 As, 71 As, 72 As, 74 As, 75 Se, 75 Br, 76 Br, 77 Br, 80 Br, 82 Br, 82 Rb, 86 Y, 88 Y, 89 Sr, 89 Zr, 97 Ru, "Tc, 110 In, m In, 113 In, 114 In, 117 Sn, 120 I, 122 Xe, 123 I, 124 I, 125 I, 166 Ho, 167 Tm, 169 Yb, 193 Pt, 195 Pt, 2O1 T1, and 203 Pb.
  • the MRI-imageable moiety can be a paramagnetic ion or a superparamagnetic particle.
  • the paramagnetic ion can be an element selected from the group consisting of Gd, Fe, Mn, Cr, Co, Ni, Cu, Pr, Nd, Yb, Tb, Dy, Ho, Er, Sm, Eu, Ti, Pa, La, Sc, V, Mo, Ru, Ce, Dy, Tl.
  • the X-ray -responsive moieties include but are not limited to iodine, barium, and barium sulfate.
  • detectable labels envisaged within the context of the present disclosure also include peptides or polypeptides that can be detected by antibody binding, e.g., by binding of a detectable labeled antibody.
  • the detectable labels are small size organic PET and SPECT labels, such as 18 F, n C or 123 I.
  • the payload is a nucleic acid.
  • the functionalized lipid particle compositions of the present application contain RNA, such as mRNA, and/or DNA.
  • the functionalized lipid particle compositions described herein comprise lipid particles that encapsulate the nucleic acid.
  • nucleic acid comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof.
  • the term comprises genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules.
  • the nucleic acid is RNA.
  • the nucleic acid is mRNA.
  • the nucleic acid is DNA.
  • the active ingredient (e.g., which is to be delivered to target cells to genetically modify the target cells and enable the target cells to express a biomolecule (such as a peptide or protein, encoded by the nucleic acid)) comprises DNA, RNA, or a mixture thereof.
  • a nucleic acid may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule.
  • a nucleic acid can be isolated.
  • isolated nucleic acid means, according to the present disclosure, that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR) for DNA or in vitro transcription (using, e.g., an RNA polymerase) for RNA, (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis.
  • PCR polymerase chain reaction
  • RNA polymerase RNA polymerase
  • nucleoside relates to compounds which can be thought of as nucleotides without a phosphate group. While a nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine. Nucleic acids may include one or more modified nucleosides or nucleotides.
  • modified nucleosides or nucleotides which may be incorporated into nucleic acids include N7-alkylguanine, N6-alkyl-adenine, 5- alkyl-cytosine, 5-alkyl-uracil, and N(l)-alkyl-uracil, such as N7-Cl-4 alkylguanine, N6-C1-4 alkyl-adenine, 5-C1-4 alkyl-cytosine, 5-C1-4 alkyl-uracil, and N(l)-Cl-4 alkyl-uracil, preferably N7-methyl-guanine, N6-methyl-adenine, 5-methyl-cytosine, 5-methyl-uridine (m5U), pseudouridine (y), and Nl-methyl-pseudouri dine (ml'P).
  • N7-alkylguanine N6-alkyl-adenine
  • 5- alkyl-cytosine 5-alkyl-cytosine
  • the nucleic acid is RNA.
  • RNA means a nucleic acid molecule which includes ribonucleotide residues. RNA typically comprises the naturally occurring nucleic acids adenosine (A), uridine (U), cytidine (C) and guanosine (G). In preferred embodiments, the RNA contains all or a majority of ribonucleotide residues.
  • ribonucleotide refers to a nucleotide with a hydroxyl group at the 2'- position of a P-D-ribofuranosyl group.
  • RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides.
  • altered/modified nucleotides can be referred to as analogs of naturally occurring nucleotides (nucleosides), and the corresponding RNAs containing such altered/modified nucleotides or nucleosides (z.e., altered/modified RNAs) can be referred to as analogs of naturally occurring RNAs.
  • a molecule contains "a majority of ribonucleotide residues" if the content of ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule.
  • the total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (z.e., naturally occurring) nucleotide residues or analogs thereof).
  • RNA includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), trans-amplifying RNA (taRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA) and immunostimulatory RNA (isRNA).
  • RNA refers to mRNA.
  • the active ingredient may be mRNA, saRNA, taRNA, or mixtures thereof.
  • the active ingredient is preferably mRNA. In some instances, the active ingredient is not siRNA.
  • the RNA comprises an open reading frame (ORF) encoding a peptide, polypeptide or protein.
  • Said RNA may capable of or configured to express the encoded peptide, polypeptide, or protein.
  • said RNA may be RNA encoding and capable of or configured for expressing a pharmaceutically active peptide or protein.
  • RNA is able to interact with the cellular translation machinery allowing translation of the peptide or protein.
  • a cell may produce the encoded peptide or protein intracellularly (e.g. in the cytoplasm), may secrete the encoded peptide or protein, or may produce it on the surface.
  • the RNA can be non-coding RNA such as antisense-RNA, micro RNA (miRNA) or siRNA.
  • miRNA micro RNA
  • siRNA siRNA
  • the nucleic acid is mRNA.
  • mRNA means "messenger-RNA” and includes a “transcript” which may be generated by using a DNA template.
  • mRNA encodes a peptide, polypeptide or protein.
  • the RNA (such as mRNA) generally contains a 5' untranslated region (5'-UTR), a peptide/polypeptide/protein coding region and a 3' untranslated region (3'-UTR).
  • mRNA is single-stranded but may contain self-complementary sequences that allow parts of the mRNA to fold and pair with itself to form double helices.
  • dsRNA means double-stranded RNA and is RNA with two partially or completely complementary strands.
  • the mRNA relates to an RNA transcript which encodes a peptide, polypeptide or protein.
  • the RNA which preferably encodes a peptide, polypeptide or protein has a length of at least 45 nucleotides (such as at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides), preferably up to 15,000, such as up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides or up to 10,000 nucleotides.
  • nucleotides such as at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000
  • the RNA (such as mRNA) is produced by in vitro transcription or chemical synthesis.
  • the RNA (such as mRNA) is produced by in vitro transcription using a DNA template.
  • IVT in vitro transcription
  • IVT does not use living/cultured cells but rather the transcription machinery extracted from cells (e.g., cell lysates or the isolated components thereof, including an RNA polymerase (preferably T7, T3 or SP6 polymerase)).
  • in vitro transcription methodology is known to the skilled person; cf., e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989.
  • in vitro transcription kits is commercially available, e.g., from Thermo Fisher Scientific (such as TranscriptAidTM T7 kit, MEGAscript® T7 kit, MAXIscript®), New England BioLabs Inc.
  • HiScribeTM T7 kit such as HiScribeTM T7 kit, HiScribeTM T7 ARCA mRNA kit
  • Promega such as RiboMAXTM, HeLaScribe®, Riboprobe® systems
  • Jena Bioscience such as SP6 or T7 transcription kits
  • Epicentre such as AmpliScribeTM
  • modified RNA such as mRNA
  • correspondingly modified nucleotides such as modified naturally occurring nucleotides, non-naturally occurring nucleotides and/or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or modifications can be effected in and/or added to the mRNA after transcription.
  • the RNA (such as mRNA) may be modified.
  • the RNA (such as mRNA) may comprise modified nucleotides or nucleosides, such as 5-methyl-cytosine, 5-methyl-uridine (m5U), pseudouridine (y) or N(l)-methyl-pseudouridine (mly).
  • the modified nucleoside may be a modified uridine.
  • the RNA may comprise a modified nucleoside in place of at least one uridine.
  • the RNA may comprise a modified nucleoside in place of each uridine (e.g., all of the uridines in the RNA are replaced with a modified nucleoside).
  • the modified nucleoside may be independently selected from pseudouridine (y), Nl-methyl-pseudouridine (mly), and 5-methyl-uridine (m5U).
  • the modified nucleoside is preferably pseudouridine ( ⁇
  • RNA such as mRNA
  • IVT-RNA in vitro transcribed RNA
  • the promoter for controlling transcription can be any promoter for any RNA polymerase.
  • RNA polymerases are the T7, T3, and SP6 RNA polymerases.
  • the in vitro transcription is controlled by a T7 or SP6 promoter.
  • a DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription.
  • the cDNA may be obtained by reverse transcription of RNA.
  • the RNA (such as mRNA) is “replicon RNA” (such as “replicon mRNA”) or simply a “replicon”, in particular "self-replicating RNA” (such as “self-replicating mRNA”) or “self-amplifying RNA” (or “self-amplifying mRNA”).
  • the lipid particles containing RNA as described herein may contain mRNA, saRNA, taRNA, or mixtures thereof.
  • the lipid particles containing RNA as described herein may contain an mRNA encoding a replicase protein, and one or more RNA molecules capable of being replicated or amplified by the replicase.
  • the nucleic acid is an inhibitory RNA.
  • inhibitory RNA means RNA which selectively hybridizes to and/or is specific for a target mRNA, thereby inhibiting (e.g., reducing) transcription and/or translation thereof.
  • Inhibitory RNA includes RNA molecules having sequences in the antisense orientation relative to the target mRNA. Suitable inhibitory oligonucleotides typically vary in length from five to several hundred nucleotides, more typically about 20 to 70 nucleotides in length or shorter, even more typically about 10 to 30 nucleotides in length. Examples of inhibitory RNA include antisense RNA, ribozyme, iRNA, siRNA and miRNA. In some embodiments of all aspects of the disclosure, the inhibitory RNA is siRNA.
  • antisense RNA refers to an RNA which hybridizes under physiological conditions to DNA comprising a particular gene or to mRNA of said gene, thereby inhibiting transcription of said gene and/or translation of said mRNA.
  • the size of the antisense RNA may vary from 15 nucleotides to 15,000, preferably 20 to 12,000, in particular 100 to 10,000, 150 to 8,000, 200 to 7,000, 250 to 6,000, 300 to 5,000 nucleotides, such as 15 to 2,000, 20 to 1,000, 25 to 800, 30 to 600, 35 to 500, 40 to 400, 45 to 300, 50 to 250, 55 to 200, 60 to 150, or 65 to 100 nucleotides.
  • small interfering RNA or "siRNA” as used herein is meant an RNA molecule, preferably greater than 10 nucleotides in length, more preferably greater than 15 nucleotides in length, and most preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length that is capable of binding specifically to a portion of a target mRNA. This binding induces a process, in which said portion of the target mRNA is cut or degraded and thereby the gene expression of said target mRNA inhibited. A range of 19 to 25 nucleotides is the most preferred size for siRNAs.
  • siRNAs comprise a single molecule in which two complementary portions are base-paired and are covalently linked by a single-stranded "hairpin” area. Without wishing to be bound by any theory, it is believed that the hairpin area of the siRNA molecule is cleaved intracellularly by the "Dicer” protein (or its equivalent) to form an siRNA of two individual base-paired RNA molecules.
  • target mRNA refers to an RNA molecule that is a target for downregulation.
  • the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide as specified herein.
  • the pharmaceutically active peptide or polypeptide is one whose expression (in particular increased expression, e.g., compared to the expression in a healthy subject) is associated with a disease.
  • the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide whose expression (in particular increased expression, e.g., compared to the expression in a healthy subject) is associated with cancer.
  • siRNA can be targeted to any stretch of approximately 19 to 25 contiguous nucleotides in any of the target mRNA sequences (the "target sequence”).
  • target sequence any of the target mRNA sequences
  • Techniques for selecting target sequences for siRNA are given, for example, in Tuschl T. et al., "The siRNA User Guide”, revised Oct. 11, 2002, the entire disclosure of which is herein incorporated by reference. Further guidance with respect to the selection of target sequences and/or the design of siRNA can be found on the webpages of Protocol Online (www.protocol-online.com) using the keyword "siRNA".
  • the sense strand of the siRNA used in the present disclosure comprises a nucleotide sequence substantially identical to any contiguous stretch of about 19 to about 25 nucleotides in the target mRNA.
  • siRNA can be obtained using a number of techniques known to those of skill in the art. For example, siRNA can be chemically synthesized or recombinantly produced. Preferably, siRNA is transcribed from recombinant circular or linear DNA plasmids using any suitable promoter. Selection of other suitable promoters is within the skill in the art. Selection of plasmids suitable for transcribing siRNA, methods for inserting nucleic acid sequences for expressing the siRNA into the plasmid, and IVT methods of in vitro transcription of said siRNA are within the skill in the art.
  • miRNA refers to non-coding RNAs which have a length of 21 to 25 (such as 21 to 23, preferably 22) nucleotides and which induce degradation and/or prevent translation of target mRNAs.
  • miRNAs are typically found in plants, animals and some viruses, wherein they are encoded by eukaryotic nuclear DNA in plants and animals and by viral DNA (in viruses whose genome is based on DNA), respectively.
  • miRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in translational repression or target degradation and gene silencing. miRNA can be obtained using a number of techniques known to those of skill in the art.
  • miRNA can be chemically synthesized or recombinantly produced using methods known in the art (e.g., by using commercially available kits such as the miRNA cDNA Synthesis Kit sold by Applied Biological Materials Inc.).
  • miRNA is transcribed from recombinant circular or linear DNA plasmids using any suitable promoter.
  • the nucleic acid is DNA.
  • DNA relates to a nucleic acid molecule which includes deoxyribonucleotide residues.
  • DNA typically comprises the naturally occurring nucleic acids adenosine (dA), thymidine (dT), cytidine (dC) and guanosine (dG) ("d” represents "deoxy”).
  • the DNA contains all or a majority of deoxyribonucleotide residues.
  • deoxyribonucleotide refers to a nucleotide which lacks a hydroxyl group at the 2'-position of a P-D-ribofuranosyl group.
  • DNA encompasses without limitation, double stranded DNA, single stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, as well as modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal DNA nucleotides or to the end(s) of DNA. It is also contemplated herein that nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present disclosure, these altered DNAs are considered analogs of naturally-occurring DNA.
  • a molecule contains "a majority of deoxyribonucleotide residues" if the content of deoxy-ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule.
  • the total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (z.e., naturally occurring) nucleotide residues or analogs thereof).
  • DNA may be recombinant DNA and may be obtained by cloning of a nucleic acid, in particular cDNA.
  • the cDNA may be obtained by reverse transcription of RNA.
  • the DNA may comprise a plasmid, a nanoplasmid, a minicircle, a transposon, or linear DNA such as doggybone DNA.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an RNA (preferably mRNA), to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (z.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of RNA (preferably mRNA) corresponding to that gene produces the protein in a cell or other biological system.
  • an RNA (such as mRNA) encodes a protein if translation of that RNA (e.g., in a cell) produces that protein.
  • the active ingredient is an RNA (preferably mRNA) or a DNA, as described in the present disclosure, which comprises a nucleic acid sequence (e.g., an ORF) encoding one or more polypeptides, e.g., a peptide or protein, preferably a pharmaceutically active peptide or protein.
  • the RNA (preferably mRNA) or DNA described in the present disclosure is capable of expressing said peptide or protein, in particular if transferred into a cell or subject.
  • the RNA (preferably mRNA) or DNA described in the present disclosure contains a coding region (ORF) encoding a peptide or protein, preferably encoding a pharmaceutically active peptide or protein.
  • RNA preferably mRNA
  • DNA encoding a pharmaceutically active peptide or protein
  • RNA preferably mRNA
  • DNA encoding a pharmaceutically active peptide or protein
  • RNA preferably mRNA
  • DNA described in the present disclosure comprises a nucleic acid sequence encoding more than one peptide or polypeptide, e.g., two, three, four or more peptides or polypeptides.
  • RNA preferably mRNA
  • DNA described in the present disclosure comprises a nucleic acid sequence encoding one or more (e.g., 1, 2, 3, 4, 5, or more) patient-specific antigens suitable for personalized cancer therapy.
  • the lipid particle compositions comprising RNA or DNA may comprise one or more species of RNA or DNA, wherein each RNA or DNA encodes a different peptide or protein.
  • the RNA (i) contains structural elements optimized for maximal efficacy of the RNA with respect to stability and translational efficiency (5' cap, 5' UTR, 3' UTR, poly(A) sequence); (ii) is modified for optimized efficacy of the RNA (e.g., increased translation efficacy, decreased immunogenicity, and/or decreased cytotoxicity) (e.g., by replacing (partially or completely, preferably completely) naturally occurring nucleosides (in particular cytidine) with synthetic nucleosides (e.g., modified nucleosides selected from the group consisting of pseudouridine (y), Nl-methyl-pseudouridine (mly), and 5-methyl-uridine); and/or codon-optimization), or (iii) both (i) and (ii).
  • (ii) is modified for optimized eff
  • pharmaceutically active peptide or protein may be understood to mean a peptide or protein that can be used in the treatment of an individual where the expression of the peptide or protein would be of benefit, e.g., in ameliorating the symptoms of a disease or disorder.
  • a pharmaceutically active peptide or protein has curative or palliative properties and may be administered to ameliorate, relieve, alleviate, reverse, delay onset of or lessen the severity of one or more symptoms of a disease or disorder.
  • a pharmaceutically active peptide or protein may have prophylactic properties and may be used to delay the onset of a disease or disorder or to lessen the severity of such disease or disorder.
  • cytokines include interferons, such as interferon-alpha (IFN-a), interferon beta (IFNP) or interferon-gamma (IFN-y), interleukins, such as interleukin 2 (IL2), IL-4, IL7, IL-10, IL-11, IL12, IL15, IL-21 and IL23, colony stimulating factors, such as colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF), erythropoietin (EPO), and bone morphogenetic protein (BMP); immunoglobulin superfamily members including antibodies (e.g., IgG), T cell receptors (TCRs), major histocompatibility complex (MHC) molecules, co-recept
  • IgG interferon-alpha
  • IFNP interferon
  • the nucleic acid encodes an antigen receptor such as a T cell receptor (TCR) or chimeric antigen receptor (CAR).
  • the pharmaceutically active peptide or protein may be or comprise a TCR or a CAR.
  • Delivering a nucleic acid encoding an antigen receptor such as a TCR or CAR to cells may be useful for generating immune effector cells genetically modified to express an antigen receptor.
  • the functionalized nucleic acid-lipid particles described herein may be used for targeted delivery of a nucleic acid encoding an antigen receptor e.g., for generating in vitro/ex vivo or in vivo immune effector cells genetically modified to express an antigen receptor.
  • the term "genetically modified”, “genetic modification” or simply “modification” includes the transfection of cells with nucleic acid.
  • transfection relates to the introduction of nucleic acids, e.g., DNA and/or RNA, into a cell.
  • 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).
  • Transfection can be transient or stable.
  • RNA or DNA can be transfected into cells to transiently express its coded protein.
  • the nucleic acid is not integrated into the nuclear genome, and will be diluted through mitosis or degraded.
  • a stable transfection is usually required for the transfected nucleic acid to enter the genome of the cell and remain in its daughter cells.
  • Such stable transfection can be achieved by using virus-based systems or transposon-based systems for transfection, for example.
  • at least a portion of transfected DNA can be inserted into the genome for stable transfection.
  • 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.
  • the functionalized particles of the present invention also contain particle forming components.
  • the one or more particle forming components comprises a cationic or cationically ionizable lipid, as defined and exemplified below.
  • such particles are lipid nanoparticles (LNP) as defined above.
  • such particles are lipoplexes (LPX) as defined above.
  • the moiety L is typically a hydrophobic moiety, such as a lipid.
  • one or more particle forming components comprises a cationic polymer, as defined and exemplified below.
  • particles formed from cationic polymers are polyplexes (PLX) as defined above.
  • the moiety L, defined below, is typically an anionic polymer, as defined and exemplified below.
  • particles formed from cationic polymers are lipidated polyplexes (LPLX), as defined above.
  • LPLX lipidated polyplexes
  • the moiety L is typically a hydrophobic moiety, such as a lipid.
  • the functionalized particles described herein also comprise a connector compound which is incorporated into the particle through a moiety incorporating the connector compound into the particle.
  • the connector compound comprises: (i) a moiety capable of incorporating the connector compound into the particle, and (ii) a first interacting moiety.
  • the moiety incorporating the connector compound into the particle is connected to a binding moiety for the docking compound (first interacting moiety) as described below.
  • the moiety incorporating the connector compound into the particle of the connector compound relates to the part of the connector compound that integrates into the particle comprising a payload (such as a nucleic acid payload).
  • the binding moiety of the connector compound relates to the part of the connector compound that forms the binding partner for the docking compound.
  • the connector 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 connector compound is covalently attached to a moiety incorporating the connector compound into the particle in a manner such that it is available for binding to the docking compound.
  • the binding moiety of the connector compound comprises a peptide or protein (e.g., an antibody or antibody fragment or a peptide tag).
  • the binding moiety of the connector 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 the connector compound into the particle.
  • a peptide or protein e.g., an antibody or antibody fragment or a peptide tag
  • the connector compound used herein comprises a moiety incorporating the connector compound into the particle which allows it to be anchored in the particle.
  • the moiety incorporating the connector compound into the particle interacts with the particle, e.g., one or more particle forming components, through electrostatic interaction (e.g., in a polyplex) or hydrophobic interaction (e.g., in an LNP).
  • the moiety incorporating the connector compound into the particle comprises a hydrophobic moiety, e.g., a lipid, such a hydrophobic lipid tail.
  • the connector compound is incorporated into the particle through a hydrophobic portion in the moiety incorporating the connector compound into the particle interacting with hydrophobic portions of the particle forming components, such as lipid bilayers, or oily lipidic cores.
  • the moiety incorporating the connector compound into the particle comprises a charged moiety, e.g., a charged polymer.
  • the connector compound is incorporated into the particle through a charge in the moiety incorporating the connector compound into the particle interacting with an opposite charge in the particle, e.g., particle forming components of the particle having an opposite charge, and/or a particle having a net opposite charge (e.g., considering all charges of the particle forming components or considering all charges of the particle forming components and the nucleic acid payload).
  • the connector compound is incorporated into the particle through a negative charge in the moiety incorporating the connector compound into the particle interacting with a positive charge of the particle.
  • the surface charge of the particle can be adjusted based on the amount and type of the connector compound, preferably based on a charged moiety of the connector compound. In embodiments, the type and/or length of the charged moiety of the connector compound is used for adjusting the surface charge.
  • a connector compound is incorporated into a particle, e.g., comprising a cationic particle forming component such as a cationic polymer, through a negative charge in the moiety incorporating the connector compound into the particle interacting with a positive charge of the particle.
  • a connector compound comprises a moiety incorporating the connector compound into the particle comprising an anionic polymer.
  • a connector compound comprises a hydrophobic component (e.g., lipid component) which allows it to integrate into (or be incorporated or anchored into) a particle comprising a particle forming component comprising a hydrophobic portion, such as a cationic or cationically ionizable lipid, or a cationic polymer comprising a hydrophobic portion, through non-covalent hydrophobic interactions, e.g., with the hydrophobic (e.g., lipidic) core of the particle.
  • a hydrophobic component e.g., lipid component
  • a connector compound may comprise a hydrophobic group (e.g., lipid), such as at least one alkyl chain, providing hydrophobic anchoring into a particle (e.g., LNP, LPX, LPLX) as described herein.
  • a hydrophobic group e.g., lipid
  • lipid such as at least one alkyl chain
  • the connector compound comprises a compound of formula (A), as described further herein, having a moiety L which has a binding moiety Bl covalently attached thereto, optionally via polymer P (when present) and/or linking moieties XI and X2.
  • a targeting compound This may also be referred to in the present specification as a “targeting compound”.
  • the connector compound is a compound of Formula (A), as defined herein.
  • the moiety L of the connector compound relates to the part of the connector compound that integrates into the particle comprising the payload.
  • the binding moiety B3 of the connector compound relates to the part of the connector compound that binds to IgD on target cells.
  • L is a hydrophobic moiety
  • L is a lipid
  • L is an anionic polymer
  • the connector compound is a compound of formula (A): L-X1-P-X2-B1 (A); wherein:
  • P is absent or comprises a polymer
  • L comprises a moiety capable of incorporating the compound into the particle, which is attached to B 1 when P is absent or to a first end of the polymer P when present; and XI and X2 are each independently absent or a linking moiety.
  • the moiety Bl is selected from the group consisting of an antibody, an antibody-like molecule, a VHH or a peptide. In one embodiment, the moiety Bl is a VHH.
  • the moiety Bl is a peptide.
  • the connector compound is non-covalently incorporated into the particle comprising the active ingredient, i.e., it forms an integral part of the particle, and the binding moiety of the connector compound is covalently attached to a hydrophobic moiety in a manner such that it is available for binding to target cells or a docking compound.
  • the binding moiety Bl of the connector compound comprises a peptide or protein (e.g., an antibody or antibody fragment or a peptide tag).
  • the binding moiety Bl of the connector 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 hydrophobic moiety (e.g., lipid).
  • the connector 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 connector 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 connector compound comprises a polymer, as defined herein, which forms the moiety P when present.
  • the hydrophobic moiety e.g., lipid
  • anionic polymer of the connector compound and the binding moiety of the connector compound are connected (typically covalently) through the polymer P.
  • the connector compound is present in an amount of 0.01 to 10 mol%, optionally 0.05 to 5 mol%, of the lipid mixture. In some embodiments, the connector compound is present in an amount of 0.1 to 2 mol%, optionally 0.1 to 1 mol%, of the lipid mixture.
  • the hydrophobic moiety comprises a lipid. In one embodiment of formula (A) or (A’), the hydrophobic moiety comprises a phospholipid. In one embodiment of formula (A) or (A’), the hydrophobic moiety comprises a moiety selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine), and mixtures thereof. In one embodiment of formula (A) or (A’), the hydrophobic moiety comprises a DSPE moiety.
  • P is absent. In one embodiment of formula (A) or (A’), P is a polymer. In one embodiment of formula (A) or (A’), P is a hydrophilic polymer. In one embodiment of formula (A) or (A’), 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
  • P comprises polyethylene glycol (PEG), preferably wherein the average molecular weight of the PEG is from about 200 to about 10,000, more preferably 500 to 5000, even more preferably 1000 to 4000, most preferably 2000.
  • PEG polyethylene glycol
  • P comprises the following general formula: wherein n is 1 to 100.
  • XI comprises a carbonyl group.
  • X2 comprises the reaction product of a thiol or cysteine reactive group with a thiol or cysteine group of a compound comprising the binding moiety B.
  • the thiol or cysteine reactive group comprises a maleimide group.
  • the hydrophobic moiety having a binding moiety covalently attached thereto comprises a distearoyl-glycero- phosphoethanolamine-polyethylene glycol-conjugate (DSPE-PEG).
  • DSPE-PEG distearoyl-glycero- phosphoethanolamine-polyethylene glycol-conjugate
  • the connector compound is a compound of formula (Al): L-X1-P-X2-B1 (Al); wherein:
  • P comprises a polymer
  • L comprises a hydrophobic moiety (e.g., lipid) or an anionic polymer attached to a first end of the polymer P;
  • Bl comprises a moiety capable of binding to B2 of the compound of formula (I) as defined below, the moiety Bl being attached to a second end of the polymer P;
  • XI is absent or a first linking moiety
  • X2 is absent or a 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.
  • L comprises DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine) which may be linked to P by an amide group.
  • P comprises a polymer as described above. In some embodiments of formula (Al), 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 poly(ethylene glycol) (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 connector 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 HS-(CH2)nC(0)-Bl, wherein n ranges from 1 to 5 and Bl comprises the binding moiety. In some embodiments, n is 2.
  • the connector compound comprises the reaction product of l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide(polyethylene glycol)] with a compound comprising the formula HS- (CH 2 )nC(O)-Bl, wherein n ranges from 1 to 5 and Bl comprises the binding moiety.
  • n is 2.
  • the connector compound is of the general formula (A2):
  • L-X1-P-X2-B1 wherein L, XI, P and B are as described above and X2 comprises a thiosuccinimide moiety.
  • the connector compound comprises the following general formula (A2’) wherein Bl comprises the binding moiety, and PEG is polyethylene glycol, as defined above (either in its broadest aspect or a preferred aspect).
  • Bl comprises a moiety comprising the structure -N-peptide-C(O)-NH2, wherein the peptide moiety is as defined herein.
  • the connector compound has the following general formula (A3): wherein P, X2 and Bl are as described above and Ri and R2 independently comprise an alkyl moiety, as defined herein (either in its broadest aspect or a preferred aspect).
  • 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 -(CH2)i7CH3 (stearyl), -(CE ⁇ isCEE (palmityl), and -(CH2)i3CH3 (myristyl).
  • the polymer P comprises poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylamino- ethoxy)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.
  • n is 8 or 14.
  • n is 14.
  • Ri and R2 in the above formula are - (CH2)i3CH3 (myristyl) and the polymer P comprises the following general formula: wherein n is 14.
  • the connector compound is of the general formula (A4): wherein P, X2 and Bl are as described above and each of Rti and Rt2 is independently H or methyl.
  • Rti and Rt2 are both methyl. In some embodiments of formula (A4), Rti is methyl, and Rt2 is H. In some embodiments of formula (A4), Rti is H, and Rt2 is methyl. In some embodiments of formula (A4), Rti and Rt2 are both H.
  • the connector compound is of the general formula (A4’): wherein P, X2 and Bl 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.
  • n is 8 or 14.
  • n is 8.
  • n is 14. In some embodiments of formula (A4), (A4’), (A4”) or (A4’”), n is 14. In some embodiments of formula (A4), (A4’), (A4”) or (A4’”), n is 14. In some embodiments of formula (A4), (A4’), (A4”) or (A4’”), n is 14.
  • the connector compound is of the general formula (A5): wherein XI, P, X2 and Bl are as described above and Ri and R2 independently comprise an acyl moiety.
  • each acyl moiety is straight or branched, preferably straight. In some embodiments of formula (A5), 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. Preferably, at least one, e.g., 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 CH3(CH2)ieC(O)- (stearoyl), CH3(CH2)i4C(O)- (palmitoyl), and CH 3 (CH 2 )i2C(O)- (myristoyl).
  • both acyl groups are CH3(CH2)ieC(O)- (stearoyl).
  • both acyl groups are CH3(CH2)i2C(O)- (myristoyl).
  • XI is absent or comprises -HPO3'(CH2)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.
  • n is 8 or 14.
  • n is 8. In some embodiments, n is 14.
  • the polymer P comprises a pSar.
  • 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 of formula (A5), s is 20 or 23.
  • the connector compound (hydrophobic moiety having a binding moiety covalently attached thereto) comprises the following general formula (A5’): wherein P, X2 and Bl are as described above and Ri and R2 independently comprise an acyl moiety.
  • 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.
  • at least one, e.g., 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 CH3(CH2)ieC(O)- (stearoyl), CH3(CH2)i4C(O)- (palmitoyl), and CH 3 (CH 2 )i2C(O)- (myristoyl).
  • both acyl groups are CH3(CH2)ieC(O)- (stearoyl).
  • both acyl groups are CH3(CH2)i2C(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.
  • n is 8 or 14.
  • n is 8.
  • n is 14.
  • n is 8 and R1 and R2 are CH3(CH2)ieC(O)- ( stearoyl). In some embodiments of formula (A5’), n is 14 and R1 and R2 are CH 3 (CH 2 )i6C(O)- (stearoyl). In some embodiments of formula (A5’), n is 8 and R1 and R2 are CH3(CH2)i2C(O)- (myristoyl). In some embodiments of formula (A5’), n is 14 and R1 and R2 are CH3(CH2)i2C(O)- (myristoyl).
  • the polymer P comprises a pSar. In some embodiments of formula (A5’), the polymer P comprises the following general 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. In some embodiments, s is 20 and R1 and R2 are CH 3 (CH 2 )i6C(O)- (stearoyl). In some embodiments, s is 20 and R1 and R2 are CH 3 (CH 2 )i2C(O)- (myristoyl).
  • X2 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 HS(CH2)nC(O)-, wherein n ranges from 1 to 5.
  • n is 2.
  • X2 comprises a thiosuccinimide moiety.
  • X2 comprises the following general formula:
  • 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 binding moiety Bl comprises an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein.
  • the connector compound is of the general formula (A10):
  • P comprises a polymer
  • L comprises a hydrophobic moiety (e.g., lipid) or an anionic polymer attached to a first end of the polymer;
  • Bl comprises an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein, attached to a second end of the polymer;
  • an epitope tag e.g., an ALFA-tag such as an ALFA-tag described herein
  • XI is absent or a first linking moiety
  • X2 is absent or a second linking moiety.
  • XI comprises a carbonyl group.
  • L comprises a phosphatidyl-ethanolamine 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.
  • a thiol or cysteine reactive group e.g., a maleimide group
  • X2 comprises a thiosuccinimide moiety.
  • L comprises a lipid as described above.
  • L comprises DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidyl-ethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine) which may be linked to P by an amide group.
  • P comprises a polymer as described above. In some embodiments of formula (A10), P comprises a polymer which provides stealth property, extends circulation half-life and/or reduces non-specific protein binding or cell adhesion. In some embodiments of formula (A10), P comprises a polymer 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) (including derivatives thereof). In some embodiments of formula (A10), P comprises polyethylene glycol (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 connector 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), with a thiol or cysteine group of a compound comprising the primary targeting moiety or epitope tag.
  • the thiol or cysteine reactive group comprises a mal eimide group.
  • the PEG reagent comprises DSPE-PEG-maleimide.
  • the compound comprising the primary targeting moiety or epitope tag comprises the formula HS(CH2)nC(O)-B, wherein n ranges from 1 to 5 and B comprises the primary targeting moiety (i.e. a moiety capable of binding to IgD) or epitope tag.
  • n is 2.
  • the connector compound comprises the reaction product of l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide(polyethylene glycol)] with a compound comprising the formula HS(CH2)nC(O)-B, wherein n ranges from 1 to 5 and B comprises the primary targeting moiety (i.e. a moiety capable of binding to IgD) or epitope tag.
  • n is 2.
  • the connector compound is of the following general formula (Al Ob): wherein Bl comprises an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein.
  • the connector compound is of the following general formula (AlOd) wherein X2 is as described above, Ri and R2 are CH3(CH2)ieC(O)- (stearoyl) or CH 3 (CH 2 )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 Bl comprises an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein.
  • ALFA-tag such as an ALFA-tag described herein.
  • n is 8 and Ri and R2 are CH3(CH2)ieC(O)- ( stearoyl). In some embodiments of formula (AlOd), n is 14 and RI and R2 are CH 3 (CH 2 )i6C(O)- (stearoyl). In some embodiments of formula AlOd) or (AlOe), n is 8 and RI and R2 are CH3(CH2)i2C(O)- (myristoyl). In some embodiments of formula (AlOd), n is 14 and RI and R2 are CH3(CH2)i2C(O)- (myristoyl). In some embodiments of formula (AlOd) X2 is of the following general formula:
  • the connector compound comprises the following general formula (AlOf): wherein X2 is as described above, Ri and R2 are CH3(CH2)ieC(O)- (stearoyl) or CH 3 (CH 2 )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 Bl comprises an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein.
  • ALFA-tag such as an ALFA-tag described herein.
  • s is 20 and Ri and R2 are CH3(CH2)ieC(O)- ( stearoyl). In some embodiments of formula (Al Of), s is 20 and Ri and R2 are CH 3 (CH 2 )i2C(O)- (myristoyl).
  • X2 comprises the following general formula:
  • Bl 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.
  • the present disclosure provides in one aspect, a connector compound as described above which is integrated in a particle (e.g., a particle as described herein) via a hydrophobic component (e.g., lipid component) of the connector compound.
  • a hydrophobic component e.g., lipid component
  • the binding moiety Bl respectively comprises a peptide or polypeptide.
  • the peptide tag comprises an ALFA- tag.
  • the connector compound is of Formula (A20):
  • P comprises a polymer
  • L comprises a hydrophobic moiety or an anionic polymer attached to a first end of the polymer
  • 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.
  • X2 comprises the reaction product of a maleimide group with a thiol or cysteine group of a compound comprising the binding moiety.
  • the hydrophobic moiety is or is comprised in a lipid.
  • the lipid comprises a phospholipid, e.g., l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).
  • the polymer provides stealth property, extends circulation half-life and/or reduces non-specific protein binding or cell adhesion.
  • the polymer comprises polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the average molecular weight of the PEG may range from 200 to 10,000, preferably 500 to 5000, more preferably 1000 to 4000, most preferably 2000.
  • the hydrophobic moiety having a binding moiety covalently attached thereto comprises a distearoyl-glycero- phosphoethanolamine-polyethylene glycol-conjugate (DSPE-PEG).
  • DSPE-PEG distearoyl-glycero- phosphoethanolamine-polyethylene glycol-conjugate
  • the binding moiety covalently attached to the hydrophobic moiety comprises a peptide, preferably the binding moiety comprises an ALFA-tag, as defined in more detail below.
  • XI and X2 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, DGlu, Asp, DAsp, Lys, DLys, hLys, DhLys, Orn, DOm, Dab, DDab, Dap, DDap, Cys, DCys, hCys, DhCys, Pen, and DPen, with the proviso that when XI is Glu, DGlu, Asp, or DAsp, X2 is Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, or DDap
  • XI is Glu and X2 is Lys.
  • K- cyclo, or -cycloE — cycloK comprises the following structure:
  • XI is Lys and X2 is Glu.
  • XI is Cys and X2 is Cys.
  • the cyclized amino acid sequence is -Ser-Arg-Leu-Glu- cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 149).
  • the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu- Arg-Lys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 150).
  • the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Lys)-Arg-Arg-Leu- Thr-Glu- (SEQ ID NO: 151).
  • the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Glu)- (SEQ ID NO: 152).
  • 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.
  • nucleic acid-lipid particles comprise connector compounds, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (Al 0), (AlOb), (AlOd), (AlOf), or (A20), such as peptide- conjugated lipids, this allows for functionalization of the nucleic acid-lipid particles.
  • the binding moiety Bl that specifically binds to the peptide of the peptide-conjugated lipid may be bound to the nucleic acid-lipid particles, wherein the binding moiety may also bind to target cells (for example by specifically binding IgD). This may provide for targeted delivery of the nucleic acid comprised within the functionalized nucleic acid-lipid particles.
  • the binding moiety that specifically binds to the peptide of the compound may be an ALFA-tag binding moiety.
  • 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 VTX1SALNAMAMG (SEQ ID NO: 145), wherein XI is I or V, the CDR2 sequence AVSX2RGNAM (SEQ ID NO: 146), wherein X2 is E, H, N, D, or S, and the CDR3 sequence LEDRVDSFHDY (SEQ ID NO: 147).
  • an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence GVTX1SALNAMAMG (SEQ ID NO: 148), 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.
  • the ALFA-tag binding moiety may comprise a single domain antibody, e.g., a camelid VHH domain comprising:
  • CDR1 - GVTISALNAMAMG SEQ ID NO: 49
  • CDR2 - AVSSRGNAM SEQ ID NO: 52
  • CDR3 - LEDRVDSFHDY SEQ ID NO: 51
  • the CDRs are provided according to the definition by AbM used by Oxford Molecular’s AbM antibody modelling software (see, generally, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer- Verlag, Heidelberg)).
  • the CDRs of the ALFA-tag binding moiety which is capable of specifically binding to an ALFA tag may also be provided according to IMGT or Kabat annotation.
  • the ALFA-tag binding moiety may comprise:
  • CDR3 - LEDRVDSFHDY (SEQ ID NO: 139); according to Kabat annotation, optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations.
  • the ALFA-tag binding moiety may comprise:
  • CDR3 - HVLEDRVDSFHDY (SEQ ID NO: 143); according to IMGT annotation, optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations.
  • the ALFA-tag binding moiety may comprise CDRs consisting of the sequences provided above.
  • the ALFA-tag binding moiety may comprise CDRs of the VHH sequence of any one of SEQ ID NO: 54-56, howsoever said CDRs are annotated.
  • an ALFA-tag binding moiety in which 1, 2 or 3 mutations have been introduced to one, two, or all three of the CDR sequences is still capable of specifically binding to the ALFA tag.
  • the ALFA-tag binding moiety may comprise or consist of the VHH sequence of any one of SEQ ID NO: 54-56 or a variant having at least 80% identity thereto.
  • the ALFA-tag binding moiety may comprise or consist of the VHH sequence of SEQ ID NO: 54 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 54.
  • the ALFA-tag binding moiety may comprise or consist of the VHH sequence of SEQ ID NO: 55 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 55.
  • the ALFA-tag binding moiety may comprise or consist of the VHH sequence of SEQ ID NO: 56 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 56.
  • an ALFA-tag binding moiety comprises a bispecific antibody which targets ALFA-tag and a cell surface antigen.
  • an ALFA-tag binding moiety comprises a moiety binding to a peptide comprising an ALFA-tag and a moiety targeting a cell surface antigen.
  • the binding moiety is a peptide
  • the compositions contain described herein may also contain a peptide-conjugated lipid.
  • peptide-conjugated lipid in its broadest sense means a lipid or lipid-like material, as defined above (either in a broadest aspect or a preferred aspect) conjugated to a peptide.
  • peptide is synonymous with “polypeptide” and “protein”.
  • the peptide comprises an ALFA-tag, (i.e., the peptide conjugated lipid may be an ALFA-conjugated lipid).
  • ALFA-tag i.e., the peptide conjugated lipid may be an ALFA-conjugated lipid.
  • the functionalized particles comprise: (a) one or more particle forming components; (b) a payload, such as a nucleic acid payload; and (c) a compound comprising: (i) a moiety capable of incorporating the connector compound into the particle, and (ii) a moiety capable of binding to immunoglobulin D (IgD), there is no connector compound as the moiety B’ of the direct binding compound (c) forms the primary targeting moiety as described below.
  • a payload such as a nucleic acid payload
  • a compound comprising: (i) a moiety capable of incorporating the connector compound into the particle, and (ii) a moiety capable of binding to immunoglobulin D (IgD)
  • IgD immunoglobulin D
  • the direct binding compound (c) is preferably a compound of Formula (A’), as defined above.
  • the compound of Formula (A’) is preferably a compound of formula (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (AlOb), (AlOd), (AlOf), or (A20), where the moiety Bl is replaced by the moiety B’.
  • the moiety B’ may take any of the preferred meanings given for Bl in the formula (Al), (AL), (A2), (A2’), (A3), (A3’), (A4), (A4’), (A5), (A5’), (A10), (AlOb), (AlOd), (AlOf), or (A20).
  • a payload such as a nucleic acid
  • a payload is delivered specifically to a target cell by providing a moiety that binds to immunoglobulin D (IgD) on target cells, e.g., an IgD antigen on target cells, thus targeting particles comprising the payload to the target cells expressing IgD on their cell surface.
  • IgD immunoglobulin D
  • the primary targeting moiety according to the present invention is a moiety capable of binding to IgD.
  • the primary targeting moiety is the moiety B’ and forms part of the direct binding compound, e.g., the compound of formula (A’).
  • the primary targeting moiety is the moiety B3 and forms part of the compound of formula (I).
  • Immunoglobulin D is an antibody isotype that makes up about 1% of proteins in the plasma membranes of immature B-lymphocytes where it is usually co-expressed with IgM. IgD is also produced in a secreted form that is found in very small amounts in blood serum, representing 0.25% of immunoglobulins in serum. The relative molecular mass and half-life of secreted IgD is 185 kDa and 2.8 days, respectively. Secreted IgD is produced as a monomeric antibody with two heavy chains of the delta (8) class, and two Ig light chains.
  • the targeting moiety that binds to IgD on target cells is comprised by a compound which is an integral part of a particle carrying the payload, i.e. the direct binding compound, e.g, the compound of formula (A’), as defined above.
  • the direct binding compound comprises a binding moiety B’ that binds to IgD on target cells.
  • the binding moiety B’ comprises a moiety binding to IgD on target cells, (e.g., a moiety binding to a cell surface IgD on target cells).
  • the moiety that binds to IgD on target cells is comprised by a compound (i.e., docking compound, e.g. compound of formula (I)) further comprising a moiety that binds to a compound (i.e., connector compound, e.g. the compound of formula (A)) which is an integral part of a particle carrying the payload and comprising a moiety for binding to the docking compound.
  • the connector compound itself preferably does not comprise a moiety that binds to IgD on target cells. Rather, the connector compound comprises a binding moiety that forms the binding partner for a docking compound which itself binds to IgD on target cells.
  • the binding moiety Bl of the connector compound is preferably a peptide tag, or a moiety binding to a peptide tag; and the moiety B2 of the docking compound comprises a moiety binding to Bl (a moiety binding to a peptide tag, or a peptide tag, respectively), and B3 of the docking compound comprises a moiety capable of binding to IgD.
  • the IgD on target cells is also referred to herein as "primary target”.
  • a "primary targeting moiety” or “moiety capable of binding to IgD” as used herein relates to the part of the connector compound or docking compound which binds to IgD, e.g., one the surface of target cells (e.g., B3 of the compound of formula (I); or B’ of the compound of formula (A’), wherein B’ or B3 is a moiety capable of binding to IgD on target cells).
  • the moiety capable of binding to IgD can be any peptide or protein (e.g. antibodies or antibody fragments) capable of binding to IgD.
  • suitable primary targeting moieties for use herein include IgD binding moieties, such as antibodies, antibody fragments and DARPins.
  • 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 may comprise: a single-chain variable fragment (scFv); a single domain binder (e.g. a VHH); an artificial binder such as a DARPin; or a singlechain derived from a T-cell receptor.
  • scFv single-chain variable fragment
  • VHH single domain binder
  • DARPin artificial binder
  • T-cell receptor a singlechain derived from a T-cell receptor
  • the antibody may be a full-length antibody, a single chain antibody fragment, a F(ab) fragment, a F(ab’)2 fragment, a F(ab’) fragment, a single domain antibody (sdAb), a nanobody, an affibody, a fibronectin artificial antibody scaffold, an anticalin, an affilin, a DARPin, a VHH, a VNAR, an iBody, an affimer, a fynomer, a domain antibody (DAb), an abdurin/ nanoantibody, a centyrin, an alphabody, or a nanofitin.
  • sdAb single domain antibody
  • the primary targeting moiety may be assessed by determining the binding affinity.
  • a quantitative assessment or measurement of binding affinity e.g. establishing a KD value
  • KD equilibrium dissociation constant
  • Ka (1/Ms) association rate constant
  • Kd dissociation rate constant
  • SPR Surface Plasmon Resonance
  • Binding affinity can also be determined using methods such as fluorescence quenching, isothermal titration calorimetry.
  • the primary targeting moiety may be capable of specifically binding to the IgD Fc region.
  • SEQ ID NO: 506 Human IgD-Fc with connecting region
  • SEQ ID NO: 507 Human IgD-Fc
  • SEQ ID NO: 508 (Rhesus IgD-Fc with connecting region)
  • the primary targeting moiety may be capable of specifically binding to one or more of SEQ ID NO: 506-509; or a variant with at least 80% sequence identity thereto.
  • the primary targeting moiety is capable of binding to SEQ ID NO: 506 or a variant with at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity thereto.
  • the primary targeting moiety is capable of binding to SEQ ID NO: 507 or a variant with at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity thereto.
  • the primary targeting moiety is capable of binding to SEQ ID NO: 508 or a variant with at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity thereto.
  • the primary targeting moiety is capable of binding to SEQ ID NO: 509 or a variant with at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity thereto.
  • the primary targeting moiety is capable of binding to each of SEQ ID NO: 506 and 508.
  • the primary targeting moiety is capable of binding to each of SEQ ID NO: 506-509.
  • the primary targeting moiety capable of binding IgD may be a single domain antibody (sdAb).
  • sdAb single domain antibody
  • the present invention provides a sdAb capable of binding IgD.
  • the sdAb may be capable of selectively binding to IgD.
  • the sdAb capable of binding IgD may comprise any features as described herein for the primary targeting moiety capable of binding IgD, wherein the primary targeting moiety is a sdAb.
  • a sdAb (i.e. a nanobody) may be defined as an antibody fragment comprising of a single monomeric variable antibody domain.
  • the sdAb may be a single chain variable domain which may be a heavy chain variable (VH) domain or light chain variable (VL) domain, having 3 CDRs.
  • VHHs variable heavy chain domain antibodies
  • SdAbs have also been engineered from heavy-chain antibodies called immunoglobulin new antigen receptor (IgNAR) found in cartilaginous fishes to produce variable new antigen receptor antibodies (VNARs).
  • IgNAR immunoglobulin new antigen receptor
  • the sdAb may be variable heavy chain domain antibody (VHH), a heavy chain variable (VH) domain, or a variable new antigen receptor antibody (VNAR).
  • VHH variable heavy chain domain antibody
  • VH heavy chain variable
  • VNAR variable new antigen receptor antibody
  • the sdAb may be a Humabody® (Crescendo Biologies).
  • a Humabody is an antibody produced by a transgenic mouse that produces heavy-chain-only antibodies with fully human VH domains, without VL domains.
  • the sdAb may be a VHH.
  • the primary targeting moiety preferably the sdAb and most preferably the VHH, may be non-human, humanised or fully human.
  • the primary targeting moiety, sdAb or VHH may be a humanised sdAb.
  • the primary targeting moiety, sdAb or VHH may be a fully human the primary targeting moiety, sdAb or VHH.
  • the remainder of the polypeptide may be any sequence which provides a suitable scaffold for the antigen binding site and displays it in an appropriate manner for it to bind the antigen.
  • a sdAb capable of binding to a ALFA tag peptide comprising CDRs comprising the following sequences:
  • the sdAb comprises SEQ ID NO: 54 or a variant with at least 80% sequence identity thereto.
  • a sdAb according to either of the above embodiments, to couple an entity of interest to an ALFA-tag, suitably wherein the ALFA-tag is incorporated into a particle, preferably a lipid nanoparticle.
  • Heavy chain variable region refers to the fragment of the heavy chain of an antigen-binding domain or antibody that contains three CDRs interposed between flanking stretches known as framework regions, which are more highly conserved than the CDRs and form a scaffold to support the CDRs.
  • Light chain variable region refers to the fragment of the light chain of an antigen-binding domain or antibody that contains three CDRs interposed between framework regions.
  • CDR complementarity determining region
  • an antigen-binding domain or antibody or antigen-binding fragment thereof refers to a highly variable loop in the variable region of the heavy chain of the light chain of an antibody. CDRs can interact with the antigen conformation and largely determine binding to the antigen (although some framework regions are known to be involved in binding).
  • the heavy chain variable region and the light chain variable region each contain 3 CDRs (heavy chain CDRs 1, 2 and 3 and light chain CDRs 1, 2 and 3, numbered from the amino to the carboxy terminus).
  • CDR-IMGT complementarity determining region
  • CDRs of the sdAbs according to the present invention may be defined using any suitable system, such as any suitable system known in the art.
  • the “Chothia-Nanobody” system is a Chothia-based numbering, with modifications to account for the structure of nanobodies/VHH.
  • VHH sequences are numbered according to the Chothia numbering scheme, with CDR1 defined as H26-H35, CDR2 defined as H51-H60 and CDR3 as H93-102.
  • Humanised antibody may refer to a genetically engineered non-human antibody, which contains human antibody constant domains and non-human variable domains modified to contain a high level of sequence homology to human variable domains. “Humanised antibody” may also refer to a sdAb which has been modified to contain a high level of sequence homology to human variable domains. This can be achieved by grafting of the three (or six) non-human antibody complementarity-determining regions (CDRs), which together form the antigen binding site, onto a homologous human acceptor framework region (FR).
  • CDRs non-human antibody complementarity-determining regions
  • FR homologous human acceptor framework region
  • Non-limiting examples of antibody humanisation methods include CDR grafting, and resurfacing (i.e.
  • a humanised antibody may comprise non-human CDR sequences, primarily human framework regions optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and, optionally, fully human constant regions.
  • additional amino acid modifications which are not necessarily back-mutations, may be introduced to obtain a humanized antibody with preferred characteristics, such as affinity and biochemical properties.
  • Humanisation of non-human therapeutic antibodies is performed to minimise its immunogenicity in man while such humanised antibodies at the same time maintain the specificity and binding affinity of the antibody of non-human origin.
  • Exemplary methods for humanisation of VHHs are described in Vincke et al. (Journal of Biological Chemistry; 2009; 284(5); 3273-3284) and Rossotti et al. (FEBS; 2021; doiilO.l 11 l/febs/15809).
  • the primary targeting moiety may comprise a combination of CDR1, CDR2 and CDR3 listed in Table 1 (see Figure 8).
  • the primary targeting moiety may comprise CDRs comprising the sequence of CDR1, CDR2 and CDR3 for a clone as listed in Table 1 ( Figure 8).
  • at least one of the CDRs listed in Table 1 may comprise one, two or three amino acid mutations.
  • the sdAb may comprise a combination of CDR1, CDR2 and CDR3 listed in Table 1 (see Figure 8).
  • the sdAb may comprise CDRs comprising the sequence of CDR1, CDR2 and CDR3 for a clone as listed in Table 1 ( Figure 8).
  • at least one of the CDRs listed in Table 1 may comprise one, two or three amino acid mutations.
  • the combination of CDR1, CDR2 and CDR3 may be selected from the CDR1, CDR2 and CDR3 for a clone as listed in Table 1 ( Figure 8); wherein each of the CDRs is defined using the same annotation system.
  • each of the CDRs may be the CDR as defined by the “Chothia-Nanobody” annotation system.
  • each of the CDRs may be the CDR as defined by the IMGT annotation system.
  • each of the CDRs may be the CDR as defined by the Kabat annotation system.
  • at least one of the CDRs listed in Table 1 may comprise one, two or three amino acid mutations.
  • the CDRs may comprise the CDR sequences shown in Table 1.
  • the CDRs may consist of the CDR sequences shown in Table 1.
  • the sdAb may comprise a CDR1 selected from the group consisting of SEQ ID NO: 1, 4, 9, 12, 15, 18, 21, 23, 25, 29, and 32, a CDR2 selected from the group consisting of SEQ ID NO: 2, 5, 7, 10, 13, 16, 19, 22 24, 26, 30, and 33; and a CDR3 selected from the group consisting of SEQ ID NO: 3, 6, 8, 11, 14, 17, 20, 27, 28, 31, and 34.
  • the primary targeting moiety or sdAb may comprise CDRs with the following sequences:
  • CDR3 - AALKLGRLGLVHMPAQYEYDY (SEQ ID NO: 3); optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations.
  • the primary targeting moiety or sdAb may comprise CDRs with the following sequences:
  • CDR1 - GFTFDDYAIG SEQ ID NO: 4
  • CDR2 - IQNKDGSTYYK SEQ ID NO: 5
  • CDR3 - AALKLGRLRLVHMPAQYEYDY SEQ ID NO: 6
  • one or more of the CDRs comprises one, two or three amino acid mutations.
  • the primary targeting moiety or sdAb may comprise CDRs with the following sequences:
  • the sdAb may comprise a CDR1 selected from SEQ ID NO: 1 and 4; a CDR2 selected from the group consisting of SEQ ID NO: 2, 5, and 7; and a CDR3 selected from the group consisting of SEQ ID NO: 3, 6, and 8.
  • the primary targeting moiety preferably a VHH or sdAb comprising CDRs with one, two or three amino acid mutations compared to the reference CDR(s) in Table 1 maintains the capacity to bind IgD.
  • any CDR defined herein by the “Chothia-Nanobody” system may be substituted with the corresponding CDR as defined by either the IMGT or Kabat system.
  • the primary targeting moiety is a VHH which comprises a sequence shown as any one of SEQ ID NO: 35-48 (see Figure 9 - Table 2) or a variant having at least 80% sequence identity thereto.
  • the present invention provides a VHH which comprises a sequence shown as any one of SEQ ID NO: 35-48 (see Figure 9 - Table 2) or a variant having at least 80% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 35 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 35 or a variant having at least 90% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 35.
  • the VHH may comprise a sequence shown as SEQ ID NO: 36 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 36 or a variant having at least 90% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 36.
  • the VHH may comprise a sequence shown as SEQ ID NO: 37 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 37 or a variant having at least 90% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 37.
  • the VHH may comprise a sequence shown as SEQ ID NO: 38 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 38 or a variant having at least 90% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 38.
  • the VHH may comprise a sequence shown as SEQ ID NO: 39 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 39 or a variant having at least 90% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 39.
  • the VHH may comprise a sequence shown as SEQ ID NO: 40 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 40 or a variant having at least 90% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 40.
  • the VHH may comprise a sequence shown as SEQ ID NO: 41 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 41 or a variant having at least 90% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 41.
  • the VHH may comprise a sequence shown as SEQ ID NO: 42 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 42 or a variant having at least 90% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 42.
  • the VHH may comprise a sequence shown as SEQ ID NO: 43 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 43 or a variant having at least 90% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 43.
  • the VHH may comprise a sequence shown as SEQ ID NO: 44 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 44 or a variant having at least 90% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 44.
  • the VHH may comprise a VHH sequence shown as SEQ ID NO: 45 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 45 or a variant having at least 90% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 45.
  • the VHH may comprise a sequence shown as SEQ ID NO: 46 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 46 or a variant having at least 90% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 46.
  • the VHH may comprise a sequence shown as SEQ ID NO: 47 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 47 or a variant having at least 90% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 47.
  • the VHH may comprise a sequence shown as SEQ ID NO: 48 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 48 or a variant having at least 90% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 48.
  • VHH refers to a polypeptide that has an equivalent function to the amino acid sequences described herein, but which includes one or more amino acid substitutions, insertions or deletions.
  • a VHH may comprise a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a sequence described herein.
  • variant is synonymous with “mutant” and refers to an amino acid sequence which differs in comparison to the corresponding wild-type sequence.
  • wild-type is used to mean a protein having an amino acid sequence respectively, which is identical with the native protein respectively.
  • Each CDR may, for example, have one, two or three amino acid mutations.
  • the VHH which comprises the IgD binding domain comprising the one or more of the CDRs which comprise one, two or three amino acid mutations may suitably maintain the capacity to bind IgD.
  • the IgD binding domain’s capacity to bind IgD may be assessed by determining the binding affinity.
  • a quantitative assessment or measurement of binding affinity e.g. establishing a KD value
  • BBI biolayer interferometry
  • Kd dissociation rate constant
  • KD equilibrium dissociation constant
  • Ka (1/Ms) association rate constant
  • Kd dissociation rate constant
  • SPR Surface Plasmon Resonance
  • Binding affinity can also be determined using methods such as fluorescence quenching, isothermal titration calorimetry.
  • the variants encompassed by the invention have a binding activity that is essentially unaltered or improved compared to the corresponding full-length sequence.
  • Identity comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % identity between two or more sequences.
  • a suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al., 1984, Nucleotide sequences Research 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid - Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching. For example, the percentage identity between two polypeptide sequences may be readily determined by BLAST which is freely available at http://blast.ncbi.nlm.nih.gov.
  • the software typically does this as part of the sequence comparison and generates a numerical result.
  • sequence may have one or more deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent molecule. These sequences are encompassed by the present invention. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the activity is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
  • the present sdAbs are capable of specifically binding to both human and rhesus macaque (Macaca mulatta) IgD. This property may be beneficial in terms of the availability of pre-clinical animal models, for example.
  • the present sdAbs may have preferential binding, or selective binding, for IgD compared to other immunoglobulins.
  • the sdAbs are capable of binding IgD but do not bind, or are essentially incapable of binding, one or more further immunoglobulins.
  • the present sdAbs may be incapable, or essentially incapable, of binding IgA, IgE, IgG and/or IgM.
  • the sdAbs may be incapable, or essentially incapable, of binding IgA, IgE, IgG and IgM.
  • the IgA, IgE, IgG and/or IgM may be human or rhesus macaque immunoglobulin.
  • the ability to bind IgD and/or the inability, or essential inability, to bind immunoglobulins other than IgD may be determined using methods which are known in the art. Methods for determining binding specificity include, but are not limited to, ELISA, western blot, immunohistochemistry, flow cytometry, Forster resonance energy transfer (FRET), phage display libraries, yeast two-hybrid screens, coimmunoprecipitation, bimolecular fluorescence complementation and tandem affinity purification.
  • FRET Forster resonance energy transfer
  • the present sdAbs may be internalised by a cell following binding to IgD on the cell surface. This may be advantageous for the delivery of a payload to a target cell, for example. Internalisation of the sdAb may be determined using methods which are known in the art, such as confocal microscropy and flow cytometry.
  • the cell may be a B cell.
  • the present sdAbs may be capable of activating a B cell following binding of the sdAb to IgD on the B cell surface. Activation of B cells may be determined using methods which are known in the art; for example, FACS analysis of known surface B cell activation markers such as CD86.
  • the present sdAb may retain the ability to bind IgD, in particular cell surface expressed IgD, in the presence of serum (e.g. human serum).
  • serum e.g. human serum
  • the ability to bind IgD in the presence of serum may be determined using methods which are known in the art, for example ELISA.
  • binding to IgD may be detectable, even in the presence of serum.
  • Retaining the ability to bind IgD in the presence of serum may mean that the level of binding to IgD is reduced by less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, less than 5% or less than 2% in the presence of serum, as compared to the level of binding to IgD in the absence of serum.
  • retaining the ability to bind IgD in the presence of serum may mean that the level of binding to IgD is reduced by less than 80%, optionally less than 50%, in the presence of serum.
  • Retaining the ability to bind IgD in the presence of serum may mean that the level of binding to IgD is at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% of the level of binding to IgD in the absence of serum.
  • retaining the ability to bind IgD in the presence of serum may mean that the level of binding to IgD is at least 10%, optionally at least 20%, of the binding to IgD detected in the absence of serum.
  • retaining the ability to binding to IgD in the presence of serum may refer to the level of binding to IgD in the presence of 50% human serum; suitably based on a 30 minute incubation at 37°C (as described in the present Examples).
  • the sdAb may be provided on the surface of a lipid nanoparticle (for example, as described in the present Examples).
  • the present invention further provides a humanised sdAb, preferably a VHH, that is capable of binding to an ALFA tag peptide.
  • humanised VHH that is capable of binding to an ALFA tag peptide may comprise CDRs comprising the following sequences:
  • one or more of the CDRs comprises one, two or three amino acid mutations.
  • the CDRs of the humanised VHH that is capable of binding to an ALFA tag peptide may also be provided according to IMGT or Kabat annotation.
  • the second binding domain may comprise CDRs comprising the following sequences:
  • CDR3 - LEDRVDSFHDY (SEQ ID NO: 139); according to Kabat annotation, optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations.
  • the second binding domain may comprise CDRs comprising the following sequences:
  • CDR3 - HVLEDRVDSFHDY (SEQ ID NO: 142); according to IMGT annotation, optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations.
  • the ALFA tag may be an ALFA tag peptide as described herein.
  • the VHH may comprise SEQ ID NO: 54 or a variant with at least 80% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 54 or a variant having at least 90% sequence identity thereto.
  • the VHH may comprise a sequence shown as SEQ ID NO: 54.
  • the variant maintains the ability to bind to an ALFA tag. Suitable variants are described herein.
  • the present invention also provides the use of a humanised sdAb as described above, preferably a VHH as described above, to couple an entity of interest to an ALFA-tag, suitably wherein the ALFA-tag is incorporated into a particle, preferably a lipid nanoparticle.
  • the present humanized sdAb may be suitable for use in a docking compound as described herein.
  • the present humanized sdAb is not restricted for use in a docking compound comprising a moiety which is capable of binding IgD.
  • the present humanized sdAb may be used in a docking compound comprising a targeting moiety capable of binding any target of interest; for example, any cell surface target of interest.
  • the functionalized particles of the present invention contain a docking compound.
  • the docking compound is a compound of formula (I), as defined herein.
  • a "docking compound” e.g.., a compound of formula (I)
  • a primary target i.e., IgD on target cells
  • a connector compound i.e., a compound of formula (A’
  • a connection between a primary target, e.g., a target cell or IgD on target cells, and a docking compound is a non-covalent connection.
  • a connection between a docking compound and a connector compound is a non-covalent or covalent connection.
  • the connector 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", as defined above, (e.g., B3 of the compound of formula (I)) e.g., a moiety targeting IgD on target cells.
  • a "primary targeting moiety” as used herein relates to the part of the docking compound which is capable of binding to or binds to IgD.
  • the docking compound further comprises a group (the moiety B2 as defined herein) which serves as a binding partner for the respective binding moiety B 1 of the connector compound of formula (A), as defined herein.
  • the portion of the connector compound comprising the hydrophobic moiety e.g., lipid
  • the moiety of the docking compound binding to the connector 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 capable of binding to IgD and a binding domain capable of binding to a connector compound.
  • the docking compound comprises an antibody or antibody fragment capable of binding to IgD and an antibody or antibody fragment capable of binding to a connector 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 capable of binding to IgD comprises a single-domain antibody such as a VHH.
  • the binding domain capable of binding to a connector compound comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody.
  • the binding domain capable of binding to a connector compound comprises a single-domain antibody such as a VHH.
  • the docking compound comprises a fusion protein which comprises a binding domain capable of binding to IgD and a binding domain capable of binding to a connector compound.
  • the docking compound comprises a single peptide chain.
  • the single peptide chain comprises a portion, e.g., antibody, antibody fragment or DARPin, capable of binding to IgD and a portion, e.g., antibody or antibody fragment, capable of binding to a connector 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 connector compound)-optional linker- VHH (a IgD) VHH (a IgD)-optional linker- VHH (a connector compound) VHH (a connector compound)-optional linker-scFv (a IgD) scFv (a IgD)-optional linker- VHH (a connector compound) VHH (a IgD)-optional linker-scFv (a connector compound) scFv (a connector compound)-optional linker- VHH (a IgD) scFv (a connector compound)-optional linker- VHH (a IgD) scFv (a connector compound)-optional linker-scFv (a IgD) scFv (a IgD)-optional linker-scFv (a connector 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 specificity binds to IgD, e.g., on target cells.
  • the specificity which binds to an epitope tag is an antibody or antibody fragment such as an NbALFA-nanobody (NbALFA).
  • NbALFA NbALFA-nanobody
  • the specificity which binds to IgD is an antibody, antibody fragment or DARPin.
  • the moiety targeting IgD is selected from the group consisting of an anti-IgD DARPin, an anti-IgDVHH and an anti-IgDscFv and/or the moiety binding to a connector 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-IgDDARPin, NbALFA x anti- IgDVHH and NbALF A x anti-IgDscFv.
  • the docking compound comprises a bispecific antibody comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-IgD 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-IgD 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-IgD DARPin.
  • the docking compound may comprise a linker which connects the B2 and B3 components.
  • Suitable linkers are well-known in the art and include, for example, serine-glycine linkers.
  • the serine-glycine linker may comprise or consist of SGGGGS (SEQ ID NO: 76) or GGGGSGGGS (SEQ ID NO: 77).
  • the docking compound may comprise or consist of the sequence of any one of SEQ ID NO: 58-74 or a variant having at least 80% identity thereto (SEQ ID NO: 58-74 are shown in Figure 10 - Table 3).
  • the docking compound may comprise or consist of the sequence of SEQ ID NO: 58 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the docking compound may comprise or consist of a sequence shown as SEQ ID NO: 58.
  • the docking compound may comprise or consist of the sequence of SEQ ID NO: 59 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the docking compound may comprise or consist of a sequence shown as SEQ ID NO: 59.
  • the docking compound may comprise or consist of the sequence of SEQ ID NO: 60 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the docking compound may comprise or consist of a sequence shown as SEQ ID NO: 60.
  • the docking compound may comprise or consist of the sequence of SEQ ID NO: 61 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the docking compound may comprise or consist of a sequence shown as SEQ ID NO: 61.
  • the docking compound may comprise or consist of the sequence of SEQ ID NO: 62 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the docking compound may comprise or consist of a sequence shown as SEQ ID NO: 62.
  • the docking compound may comprise or consist of the sequence of SEQ ID NO: 63 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the docking compound may comprise or consist of a sequence shown as SEQ ID NO: 63.
  • the docking compound may comprise or consist of the sequence of SEQ ID NO: 64 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the docking compound may comprise or consist of a sequence shown as SEQ ID NO: 64.
  • the docking compound may comprise or consist of the sequence of SEQ ID NO: 65 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the docking compound may comprise or consist of a sequence shown as SEQ ID NO: 65.
  • the docking compound may comprise or consist of the sequence of SEQ ID NO: 66 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the docking compound may comprise or consist of a sequence shown as SEQ ID NO: 66.
  • the docking compound may comprise or consist of the sequence of SEQ ID NO: 67 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the docking compound may comprise or consist of a sequence shown as SEQ ID NO: 67.
  • the docking compound may comprise or consist of the sequence of SEQ ID NO: 68 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the docking compound may comprise or consist of a sequence shown as SEQ ID NO: 68.
  • the docking compound may comprise or consist of the sequence of SEQ ID NO: 69 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the docking compound may comprise or consist of a sequence shown as SEQ ID NO: 69.
  • the docking compound may comprise or consist of the sequence of SEQ ID NO: 70 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the docking compound may comprise or consist of a sequence shown as SEQ ID NO: 70.
  • the docking compound may comprise or consist of the sequence of SEQ ID NO: 71 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the docking compound may comprise or consist of a sequence shown as SEQ ID NO: 71.
  • the docking compound may comprise or consist of the sequence of SEQ ID NO: 72 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the docking compound may comprise or consist of a sequence shown as SEQ ID NO: 72.
  • the docking compound may comprise or consist of the sequence of SEQ ID NO: 73 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the docking compound may comprise or consist of a sequence shown as SEQ ID NO: 73.
  • the docking compound may comprise or consist of the sequence of SEQ ID NO: 74 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.
  • the docking compound may comprise or consist of a sequence shown as SEQ ID NO: 74.
  • the moiety on the connector compound e.g. moiety Bl of the compound of formula (A), binding moiety covalently attached to a hydrophobic moiety or an anionic polymer
  • the moiety on the docking compound e.g. moiety B2 of the compound of formula (I), moiety binding to the binding moiety covalently attached to a hydrophobic moiety or anionic polymer
  • interacting with each other e.g., non-covalently bind to each other.
  • the moieties on the connector compound and on the docking compound interacting with each other bind to each other under physiological conditions.
  • the moieties on the connector compound and on the docking compound interacting with each other are antibody/antigen systems.
  • the moiety of the connector compound binding to the docking compound comprises a peptide or protein, e.g., a peptide tag
  • the moiety of the docking compound binding to the connector 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 connector compound comprises a peptide or protein, e.g., a peptide tag
  • the moiety of the connector compound binding to the docking compound comprises a binder, e.g., an antibody or antibody fragment, binding to the peptide or protein.
  • moiety Bl comprises an antibody, an antibody-like molecule, or a VHH, and moiety B2 is a peptide.
  • moiety B2 comprises an antibody, an antibody-like molecule, or a VHH, and moiety Bl is a peptide.
  • the moiety Bl of the compound of formula (A) comprises a peptide tag
  • the moiety B2 of the compound of formula (I) binds to the compound of formula (A) via the peptide tag.
  • the peptide tag comprises an ALFA-tag.
  • the moiety B2 of the compound of formula (I) is a VHH capable of binding to the ALFA-tag.
  • the moiety B2 of the compound of formula (I) comprises a peptide tag
  • the moiety Bl of the compound of formula (A) binds to the compound of formula (I) via the peptide tag.
  • the peptide tag comprises an ALFA-tag.
  • the moiety Bl of the compound of formula (A) is a VHH capable of binding to the ALFA-tag.
  • the moieties on the connector compound and on the docking compound interacting with each other comprise an epitope tag/binder system.
  • 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.
  • the epitope tag/binder system comprises an ALFA-tag and an ALFA-specific single-domain antibody (sdAb), NbALFA-nanobody.
  • sdAb single-domain antibody
  • the ALFA-tag may be defined as described below.
  • the functionalized particles of the present invention comprise polymers.
  • polymer 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.
  • 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.
  • 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.
  • the particle forming components may comprise 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).
  • the polymer is a hydrophilic polymer and the connector 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.
  • the polymer component faces the outside of said particle and forms a protective hydrophilic shell surrounding the particle.
  • the polymer portion of the amphiphilic derivative contributes to conferring stealth properties on the particles.
  • the polymer portion of the amphiphilic derivative confers 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 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.
  • the polymer 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) (including derivatives thereof), as defined and exemplified below.
  • PEG poly(ethylene 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 polymer is PEG
  • the PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide.
  • the 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 moiety of the amphiphilic derivative of a polymer has a molecular weight of 1000 or more.
  • the PEG moiety of the amphiphilic derivative of a polymer comprises 2 units or more, such as 5 units of more, such as 10 units or more of formula (O-CH2-CH2) n (where n is the number of ethylene oxide units). In some embodiments, 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-PEG5000 are used as the amphiphilic derivative of a polymer.
  • the polymer is a pSar and the 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 polymer is POX and/or POZ
  • 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 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: wherein Rn is as defined above.
  • the POX and/or POZ polymer is a polymer of POZ and comprises repeating units of the following general formula: wherein Rn is as defined above.
  • m i.e., the number of repeating units in the polymer
  • m 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 110 between 10 and 100, between 10 and 90, between 10 and 80, or between
  • 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 Rn is as defined above.
  • Rn is as defined above.
  • 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.
  • 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.
  • Rn at each occurrence may be the same alkyl group (e.g., Rn may be methyl in each repeating unit).
  • Rn in at least one repeating unit differs from Rn in another repeating unit (e.g., for at least one repeating unit Rn is one specific alkyl (such as ethyl), and for at least one different repeating unit Rn is a different specific alkyl (such as methyl)).
  • each Rn may be selected from two different alkyl groups (such as methyl and ethyl) and not all Rn are the same alkyl.
  • Rn preferably is methyl or ethyl, more preferably methyl.
  • each Rn is methyl or each Rn is ethyl.
  • Rn is independently selected from methyl and ethyl for each repeating unit, wherein in at least one repeating unit Rn is methyl, and in at least one repeating unit Rn 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, as defined herein.
  • pAEEA poly-2-(2-(2-aminoethoxy)ethoxy)- acetic acid
  • pMAEEA poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid
  • the polymer comprises the following general formula: wherein
  • X 11 and X 12 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 11 is -C(O)- and X 12 is -NR 1 -, wherein R 1 is hydrogen or Ci-8 alkyl. In some embodiments, X 11 is -C(O)- and X 12 is -NR 1 -, wherein R 1 is hydrogen or methyl. In some embodiments, X 11 is -C(O)- and X 12 is -NR 1 -, wherein R 1 is hydrogen. In some embodiments, Y is -CH2- or -(CIfc)?-. In some embodiments, Y is -CH2-.
  • the polymer comprises the following general formula: wherein
  • R 1 is hydrogen or C1-8 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 C1-8 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. In some embodiments, 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. In some embodiments, the molar proportion of the amphiphilic derivative of a polymer integrated 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 functionalized particles of the present invention comprise a cationic polymer, as particle forming component.
  • a cationic polymer means a polymer, as defined generally herein, which carries at least one cation, i.e. an ionic species having a positive charge.
  • 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.
  • a connector compound is incorporated into the particle comprising a cationic polymer through a negative charge in the moiety incorporating the connector compound into the particle interacting with a positive charge of the particle.
  • a connector compound is incorporated into the particle comprising a cationic polymer through a moiety incorporating the connector compound into the particle comprising an anionic polymer.
  • the cationic polymer comprises one or more selected from the group consisting of cationic or polycationic peptides or proteins, including protamine, spermin or spermidine, poly-lysine, poly-arginine, cationic polysaccharides, including chitosan, cationic polymers, including poly(ethyleneimine), poly(propyleneimine), polybrene, polyallylamines, and polyvinylamine.
  • the polymer comprises a polyamidoamine (PAMAM) polymer.
  • a cationic polymer is a homopolymer selected from poly(ethylenimine), poly(propylenimine), polybrene, polyallylamine, polyvinylamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L-histidine, and poly(2- aminoethyl methacrylate), or a pharmaceutically acceptable salt thereof.
  • a cationic polymer is linear.
  • a cationic polymer is a linear polymer selected from poly(ethylenimine), poly(propylenimine), polybrene, polyallylamine, polyvinylamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L-histidine, and poly(2-aminoethyl methacrylate).
  • a cationic polymer is a branched polymer selected from poly(ethylenimine), poly(propylenimine), polybrene, polyallylamine, polyvinylamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L-histidine, and poly(2-aminoethyl methacrylate).
  • the cationic polymer comprises poly(ethyleneimine).
  • the poly(ethyleneimine) is a linear polymer.
  • the poly(ethyleneimine) is a branched polymer.
  • the poly(ethyleneimine) has a mean molar mass between 1000 Da and 150000 Da, between 5000 Da and 100000 Da, between 10000 Da and 50000 Da, between 15000 Da and 30000 Da, between 20000 Da and 25000 Da, or of about 22500 Da.
  • the poly(ethyleneimine) has a mean molar mass between 22500 Da and 150000 Da.
  • the functionalized particles of the present invention comprise a polyamine derivative, e.g., a carboxylated polyamine derivative, as particle forming component.
  • 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.
  • the polyamine derivative has a linear poly(ethyleneimine) 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 poly(ethyleneimine) 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.
  • the particle forming components comprise a compound comprising the following formula:
  • the 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.
  • the functionalized particles of the present disclosure especially when in the form of a polyplex (PLX), contain an anionic polymer.
  • anionic polymer means a polymer, as defined generally herein, which carries at least one anion, i.e. an ionic species having a negative charge.
  • an anionic polymer described herein can be linear or branched, and comprises one or more anionic moieties or groups.
  • an anionic polymer is a polyanionic polymer, e.g., a polymer having one or more anionic groups.
  • an anionic group is a -CCh', a -OSCh', or a -OPCh 2 ' group.
  • the anionic polymer is a homopolymer.
  • the anionic polymer is a heteropolymer.
  • an anionic polymer is polyglutamic acid. In some embodiments, an anionic polymer is poly-L-glutamic acid. In some embodiments, an anionic polymer is poly aspartic acid. In some embodiments, an anionic polymer is poly-L-aspartic acid. In some embodiments, an anionic polymer is a polyphosphate. In some embodiments, an anionic polymer is a homopolymer. In some embodiments, an anionic polymer is a homopolymer comprising about 10 to about 150 repeating monomeric units. In some embodiments, an anionic polymer is a homopolymer comprising about 10 to about 100 repeating monomeric units.
  • an anionic polymer is a homopolymer comprising about 20 to about 100 repeating monomeric units. In some embodiments, an anionic polymer is a homopolymer comprising about 20 to about 80 repeating monomeric units. In some embodiments, an anionic polymer is a homopolymer comprising about 50 repeating monomeric units. In some embodiments, an anionic polymer is a homopolymer comprising about 100 repeating monomeric units.
  • an anionic polymer is a poly-L-glutamic acid homopolymer comprising about 10 to about 150 repeating units of glutamic acid. In some embodiments, an anionic polymer is a poly-L-glutamic acid homopolymer comprising about 10 to about 100 repeating units of glutamic acid. In some embodiments, an anionic polymer is a poly-L-glutamic acid homopolymer comprising about 20 to about 100 repeating units of glutamic acid. In some embodiments, an anionic polymer is a poly-L-glutamic acid homopolymer comprising about 20 to about 80 repeating units of glutamic acid.
  • an anionic polymer is a poly-L- glutamic acid homopolymer comprising about 50 repeating units of glutamic acid. In some embodiments, an anionic polymer is a poly-L-glutamic acid homopolymer comprising about 100 repeating units of glutamic acid.
  • compositions such as the aqueous dispersions, nucleic acid-lipid particles and functionalized nucleic acid-lipid particles
  • lipid and “lipid-like material” are broadly defined herein as molecules which comprise one or more hydrophobic moieties or groups and also one or more hydrophilic moieties or groups. 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.
  • Lipids may comprise a polar portion and an apolar (or non-polar) portion.
  • amphiphile as used in this specification is broadly defined herein as a molecule comprising hydrophobic moieties and hydrophilic moieties and/or a polar and apolar portion. As both cationic and anionic lipids both contain such groups, they are therefore amphiphiles. In this specification the term “cationic lipid” is therefore synonymous with “cationic amphiphile” and the term “anionic lipid” is synonymous with “anionic amphiphile”.
  • Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated hydrocarbyl groups (as defined and exemplified above), such as alkyl, alkenyl and/or alkynyl groups and such groups substituted by one or more aryl, heteroaryl, or cycloalkyl groups (as defined and exemplified above).
  • apolar groups that include, but are not limited to, long-chain saturated and unsaturated hydrocarbyl groups (as defined and exemplified above), such as alkyl, alkenyl and/or alkynyl groups and such groups substituted by one or more aryl, heteroaryl, or cycloalkyl groups (as defined and exemplified above).
  • the hydrophilic groups may comprise polar and/or charged groups and include at least one amine and optionally hydrophilic non-charged groups such as hydroxyl, carbohydrate, sulfhydryl, nitro or like groups and may further include anionic groups such as phosphate, phosphonate, carboxylic acid, sulfate, sulfonate (all as defined and exemplified above) and other like groups.
  • hydrophobic as used herein with respect to a compound, group or moiety means that said compound, group, or moiety is not attracted to water molecules and, when present in an aqueous solution, excludes water molecules.
  • hydrophobic refers to any compound, group or moiety which is substantially immiscible or insoluble in aqueous solution.
  • a hydrophobic compound, group or moiety is substantially nonpolar.
  • hydrophobic groups are hydrocarbyl groups (as defined and exemplified above), such as alkyl, alkenyl and/or alkynyl groups and such groups substituted by one or more aryl, heteroaryl, or cycloalkyl groups (as defined and exemplified above).
  • the hydrophobic group can have functional groups (e.g., ether, thioether, ester, dioxolane, halide, amide, sulfonamide, carbamate, 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.
  • the hydrophobic moieties of a lipid may have between 24 and 60 carbon atoms and can be hydrocarbyls (as described and exemplified above, typically comprising alkyl, alkenyl or alkynyl groups as described and exemplified above).
  • the 24 to 60 carbon atoms can be segmented into two or more hydrophobic moieties, with each such moiety typically having at least 6 carbon atoms.
  • An example for segmented hydrophobic moieties wherein each segment is hydrocarbyl are lipids comprising the DACA moiety as described in WO2011/003834 wherein each of the acyl or alkyl groups comprise between 12 and 20 carbon atoms.
  • Another example are lipids wherein the hydrophobic moiety comprises a steroid moiety, such as a cholesteryl moiety.
  • the hydrophobic moieties of a lipid preferably have between 24 and 60 carbon atoms and can also be heterohydrocarbyls wherein the heteroatoms are selected from N, O or S forming one, two, three or four non-charged groups of ether, thioether, ester, amide, carbamate, sulfonamide and the like.
  • the 24 to 60 carbon atoms can be segmented into two or more hydrophobic moieties, provided that each such moiety has at least 6 carbon atoms.
  • segmented hydrophobic moieties wherein each segment is hydrocarbyl are lipids comprising the diacylglycerol or dialkylglycerol moiety wherein each of the acyl or alkyl comprise between 12 and 20 carbon atoms.
  • hydrophobic moieties wherein each segment is heterohydrocarbyl are the ester-branched moieties in lipids such as SM-102 or ALC-315, as defined and exemplified below.
  • compositions (such as the aqueous dispersions, nucleic acid-lipid particles and functionalized nucleic acid-lipid particles) of the present invention also contain a cationic lipid or cationically ionizable lipid, or a mixture of any thereof.
  • cationic lipid means a lipid or lipid-like material, as defined herein, having a constitutive positive charge.
  • a “constitutive charge” means that the cationic lipid carries the positive charge at all physiological pH.
  • the cationic lipids carrying constitutive charged cationic moieties are typically quaternary ammonium salts (as defined above) or salts of organic bases, such as nitrogen-containing bases.
  • organic bases are strong bases (i.e. bases which are completely protonated when dissolved in a solvent, such as but not limited to an aqueous solvent, such that the concentration of the unprotonated species is too low to be measured).
  • the cationic lipid is a monovalent cationic lipid.
  • the cationic lipid contains a charged polar moiety selected from the group consisting of guanidinium, ammonium, imidazolium, pyridinium, amidinium, and piperazinium.
  • cationic lipids include, but are not limited to l,2-dialkyloxy-3- dimethylammonium propanes and l,2-dialkenyloxy-3 -dimethylammonium propanes (each alkyl or alkenyl portion being as defined and exemplified above and preferably having 12 to 20 carbon atoms), such as l,2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA), l,2-diacyloxy-3 -dimethylammonium propanes (the alkyl or alkenyl part of each acyl portion being as defined and exemplified above and preferably having 12 to 20 carbon atoms), such as l,2-dioleoyl-3 -trimethylammonium propane (DOTAP) or l,2-dioleoyl-3 -dimethylammonium -propane (DODAP); dimethyldioctadecylammonium (DDAB);
  • DODMA and DODAP are shown below.
  • DOTMA DOTMA
  • DOTAP DOTAP
  • analogues thereof are shown below.
  • DOTAP and further suitable homologues are shown below.
  • Further suitable cationic lipids are described in Sun and Lu, Pharmaceutical Research, 2023, https://doi.org/10.1007/sl l095-022-03460-2.
  • the lipid is a cationically ionizable lipid.
  • a "cationically ionizable lipid” refers to a lipid or lipid-like material which, depending on whether it is protonated or deprotonated, has a net positive charge or is neutral, i.e., a lipid which is not permanently cationic.
  • the cationically ionizable lipid is either positively charged or neutral.
  • the cationically ionizable lipid comprises a head group which includes at least one nitrogen atom (N) which is capable of being protonated, preferably under physiological or slightly acidic conditions.
  • the cationic or cationically ionizable lipid is a compound represented by formula (TL-I):
  • L 1 and L 2 are each independently an optionally substituted C1-C30 aliphatic group
  • L 3 is a bond, optionally substituted C1-C10 aliphatic group, or optionally substituted 2- to 10-membered heteroaliphatic group;
  • X 1 and X 2 are each independently selected from a bond, -OC(O)-, -C(O)O-, - S(O) 2 N(R 1 )-, -N(R 1 )S(O)2, -S(O)-, -S(O) 2 -, -S(O) 2 C(R 1 ) 2 -, -OC(S)C(R 1 ) 2 -, - C(R 1 ) 2 C(S)O-, and -S-, wherein one or both of X 1 or X 2 is selected from - S(O) 2 N(R 1 )-, -N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O) 2 C(R 1 )2-, -OC(S)C(R 1 )2-, - C(R 1 ) 2 C(S)O-, and -S-; each R 1 is, independently, at each
  • T 1 and T 2 are each independently an optionally substituted C3-C30 aliphatic;
  • R 4 is optionally substituted 4- to 12-membered heterocycle, optionally substituted 4- to 12 membered heteroaryl, Ce-Ci 2 aryl substituted with one or more of-(CH 2 )o- 6-OH or -(CH 2 )O-6-N(R 5 ) 2 , or Cs-Cn cycloaliphatic substituted with one or more of oxo, -(CH 2 )O-6-OH, or -(CH 2 )O-6-N(R 5 ) 2 ; each R 5 is independently selected from H and optionally substituted Ci-Ce aliphatic.
  • L 1 and L 2 are each independently -(CH 2 )e- io-.
  • X 1 and X 2 are each independently selected from a -S(0) 2 N(R 1 )-, -N(R 1 )S(0) 2 , -S(0)-, -S(0) 2 -, -S(O) 2 C(R 1 ) 2 -, -OC(S)C(R 1 ) 2 -, - QR ⁇ C S) ⁇ -, and -S-.
  • X 1 and X 2 are each -S(O) 2 N(R 1 )-, where each R 1 is independently R 1 is Ci-Cio aliphatic.
  • T 1 and T 2 are each independently selected from optionally substituted C3-C 2 o alkyl.
  • T 1 and T 2 are each independently selected from:
  • G is -N(R 2 )C(S)N(R 2 )2 or -N(R 5 )S(O)2R 3 . In some embodiments of formula (TL-I), G is -N(H)C(S)N(R 2 )2, where each R 2 is selected from optionally substituted Ci-Ce aliphatic and OH.
  • G is -OH. In some embodiments of formula (TL-I), G is selected from:
  • -L 3 -G is selected from:
  • the compound is represented by Formula (TL-IIa):
  • TL-IIa or a pharmaceutically acceptable salt thereof.
  • the compound is represented by Formula (TL-IIc): TL-IIc or a pharmaceutically acceptable salt thereof.
  • the compound is represented by Formula (TL-IIIb):
  • TL-IIIb or a pharmaceutically acceptable salt thereof.
  • the compound is represented by Formula (TL-IIIe):
  • the compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-I)
  • the compound is 7,7’ -((4- hydroxybutyl)azanediyl)bis(N-hexyl-N-octylheptane-l-sulfonamide) or a pharmaceutically acceptable salt thereof.
  • the compound is 7,7’-((4-(3,3- dimethylthioureido)butyl)azanediyl)bis(N-hexyl-N-octylheptane-l-sulfonamide) or a pharmaceutically acceptable salt thereof.
  • a tail-linker moiety is any bivalent linker, such as an aliphatic or heteroaliphatic group; a tail-end is a hydrophobic group, e.g., an aliphatic group, a head group is a polar or cationic or ionizable head group, a tail junction is a biodegradable group, such as an ester, or a sulfur-containing moiety (e.g., thioether, sulfonyl, or sulfonamide), and a head-tail junction is a central atom or functional group connecting a tail or tails to the head group (e.g., a tertiary amine group).
  • a tail-end is a hydrophobic group, e.g., an aliphatic group
  • a head group is a polar or cationic or ionizable head group
  • a tail junction is a biodegradable group, such as an ester, or a sulfur-containing moiety
  • TL-IV or a pharmaceutically acceptable salt thereof, the method comprising: contacting a compound represented by Formula (TL-V)
  • TL-V with a compound represented by one of Formulae (TLVIa)-(TL-VIc)
  • each of L 4 and L 5 are each independently an optionally substituted C1-C30 aliphatic group;
  • L 6 is a bond, optionally substituted C1-C10 aliphatic group, or optionally substituted 2- to 10-membered heteroaliphatic group;
  • X 3 and X 4 are each independently selected from a bond, -OC(O)-, -C(O)O-, - S(O) 2 N(R 40 )-, -N(R 40 )S(O)2, -S(O)-, -S(O) 2 -, -S(O) 2 C(R 40 )2-, -OC(S)C(R 40 )2-, - C(R 40 )2C(S)O-, or -S-, wherein one or both of X 3 or X 4 is selected from - S(O) 2 N(R 40 )-, -N(R 40 )S(O)2, -S(O)-, -S(O) 2 C(R 40 )2-, -OC(S)C(R 40 )2-, - C(R 40 )O-, or -S-; each R 40 is, independently, at each instance, optional
  • T 3 and T 4 are each independently an optionally substituted C3-C20 aliphatic;
  • G 1 is -N(R 6 )C(S)N(R 6 ) 2 , -OH, -N(R 6 ) 2 , -N(R 9 )C(O)R 7 , -N(R 9 )S(O) 2 R 7 , - N(R 9 )C(O)N(R 7 ) 2 , -CH(N-R 7 ), or-R 8 ;
  • each G 2 is independently O or N2;
  • each G 3 is independently halogen (e.g., Cl, Br, or I), -OTs, or OTf;
  • each R 6 is, independently, at each instance, selected from the group consisting of H, optionally substituted Ci-Ce aliphatic or OR 7 ; or two instances of R 6 come together with the atoms to which they are attached to form an optionally substituted 4- to 12-membered heterocycle ring or an optionally substituted 4- to 12-membered heteroaryl ring;
  • each R 7 is, independently
  • R 8 is optionally substituted 4- to 12-membered heterocycle, optionally substituted 4- to 12 membered heteroaryl, C6-C12 aryl substituted with one or more of-(CH2)o-6- or C3-C12 cycloaliphatic substituted with one or more of oxo, -(CH 2 )O-6-OH, or -(CH 2 )O-6-N(R 9 ) 2 ; and each R 9 is independently selected from H and optionally substituted Ci-Ce aliphatic.
  • a reducing agent is NaBFFCN or NaBH(OCOCH3)3. In some embodiments, a reducing agent is NaBFLCN. In some embodiments, a reducing agent is NaBH(OCOCH 3 )3.
  • a method of preparing a compound as described herein further comprises preparing a compound represented by Formula (TL-VIa) or the compound represented by Formula (TL-VIIa), wherein G 1 is O,
  • TL-VIa TL-VIIa by contacting a compound represented by Formula (TL-VIII) or a compound represented by Formula (TL-IX):
  • an oxidizing agent is DMSO, PCC, or DMP. In some embodiments, an oxidizing agent is DMSO, and the method further comprises contacting the compound represented by Formula VIII or IX and DMSO with a sulfur trioxide pyridine complex (SOs’pyridine).
  • SOs sulfur trioxide pyridine complex
  • a method of preparing a compound described herein further comprises preparing the compound represented by Formula (TL-VIII) or (TL-IX):
  • TL-VIII TL-IX by contacting a compound represented by Formula (TL-X) or a compound represented by Formula (TL-XI): with a compound represented by Formula (TL-XII) or a compound represented by Formula (TL-XIII):
  • the cationic or cationically ionizable lipid is selected from the group consisting of:
  • DODMA 1.2-dioleoyloxy-3 -dimethylaminopropane
  • MPDACA 4-methylpyridinium-di(heptadecyl)methylcarboxylic acid
  • DOTAP 1.2-dioleoyl-3 trimethylammonium propane
  • DODAP 1.2-dioleoyl-3-dimethylammomium propane
  • DOTMA 1.2-di-O-octadecenyl-3 -trimethylammonium propane
  • BODD-C2C4-PipZ bis(2-octyldodecyl) 3,3'-((4-(4-methylpiperazin-l- yl)butyl)azanediyl)dipropionate
  • BODD-C2C2-DMA bis(2-octyldodecyl) 3,3'-((2- (dimethylamino)ethyl)azanediyl)dipropionate
  • BODD-C2C2-lMe-Pyr bis(2-octyldodecyl) 3,3'-((2-(l-methylpyrrolidin-2- yl)ethyl)azanediyl)dipropionate
  • BODD-C2C2-Pyr bis(2-octyldodecyl) 3,3'-((2-(pyrrolidin-l- yl)ethyl)azanediyl)dipropionate) or a mixture of any thereof.
  • the cationic lipid is palmitoyl-oleoyl-nor-arginine (PONA). In one embodiment, the cationic lipid is 4-methylpyridinium-di(heptadecyl)- methylcarboxylic acid (MPDACA). In one embodiment, the cationic lipid is 1,2- di oleoyloxy-3 -trimethylammonium propane (DOTAP). In one embodiment, the cationic lipid is l,2-dioleoyl-3 -dimethylammonium -propane (DODAP).
  • PONA palmitoyl-oleoyl-nor-arginine
  • MPDACA 4-methylpyridinium-di(heptadecyl)- methylcarboxylic acid
  • DOTAP 1,2- di oleoyloxy-3 -trimethylammonium propane
  • DODAP l,2-dioleoyl-3 -dimethylammonium -propane
  • the cationically ionizable lipid is 7,7’-((4-hydroxybutyl)- azanediyl)bis(N-hexyl-N-octylheptane-l -sulfonamide) (BNT-51). In one embodiment, the cationically ionizable lipid is [(4-hydroxybutyl)azanediyl]- di(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC-315). In one embodiment, the cationically ionizable lipid is 1,2-di oleoyloxy-3 -dimethylaminopropane (DODMA).
  • DODMA 1,2-di oleoyloxy-3 -dimethylaminopropane
  • the cationically ionizable lipid is 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA). In one embodiment, the cationically ionizable lipid is heptatriaconta-6,9,28,31-tetraen-19-yl-4- (dimethylamino)butanoate (D-Lin-MC3-DMA).
  • the cationically ionizable lipid is l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA)/ In one embodiment, the cationically ionizable lipid is di((Z)-non-2-en-l-yl)-9-((4- (dimethylaminobutanoyl)oxy)heptadecanedioate (L319). In one embodiment, the cationically ionizable lipid is A/.s-(2-butyloctyl) 10-(N-(3-(dimethylamino)propyl)- nonanamido)-nonadecanedioate (A9).
  • the cationically ionizable lipid is (heptadecan-9-yl 8- ⁇ (2-hydroxyethyl)[6-oxo-6-(undecyloxy)octyl]amino ⁇ - octanoate) (L5). In one embodiment, the cationically ionizable lipid is heptadecan-9- yl 8- ⁇ (2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino ⁇ -octanoate) (SM-102).
  • the cationically ionizable lipid is O-[N- ⁇ (9Z,12Z)-octadeca-9,12- dien-l-yl) ⁇ -N- ⁇ 7-pentadecylcarbonyloxyoctyl ⁇ -amino]4-(dimethylamino)butanoate (HY501).
  • the cationically ionizable lipid is 2-(di-((9Z,12Z)- octadeca-9,12-dien-l-yl)amino)ethyl 4-(dimethylamino)butanoate (EA-2).
  • the cationically ionizable lipid is BODD-C2C4-PipZ. In one embodiment, the cationically ionizable lipid is BHD-C2C2-PipZ. In one embodiment, the cationically ionizable lipid is BODD-C2C2-DMA. In one embodiment, the cationically ionizable lipid is BODD-C2C2-lMe-Pyr. In one embodiment, the cationically ionizable lipid is BODD-C2C2-Pyr.
  • the cationically ionizable lipid is selected from those described generally and specifically in WO 2018/087753. In some embodiments, the cationically ionizable lipid is selected from the group consisting of:
  • Hy 501 m.w: 761.26
  • the cationically ionizable lipid is 4-((di-((9Z,12Z)-octadeca-9,12- dien-l-yl)amino)oxy)-7V,7V-dimethyl-4-oxobutan-4-amine (HYAM-2).
  • the cationically ionizable lipid is ((2-(4-(dimethylamino)butanoyl)- oxy)ethyl)-azanediylbis(octane 8,1 -diyl) bis(2-hexyldecanoate) (EA-405).
  • the cationically ionizable lipid is (2-(4-(dimethylamino)butanoyl)- oxy)azanediylbis-(octane 8, 1 -diyl) bis(2-hexyldecanoate) (HY-405).
  • the cationically ionizable lipid is O-[N- ⁇ (9Z,12Z)-octadeca-9,12-dien-l- yl) ⁇ -N- ⁇ 7-pentadecylcarbonyloxyoctyl ⁇ -amino]4-(dimethylamino)butanoate (HY501).
  • the cationic or cationically ionisable lipid is present in an amount of 20 to 70 mol% of the total lipids present in the lipid mixture. In one embodiment, the cationic or cationically ionisable lipid is present in an amount of 30 to 60 mol% of the total lipids present in the lipid mixture. In one embodiment, the cationic or cationically ionisable lipid is present in an amount of 40 to 50 mol% of the total lipids present in the lipid mixture.
  • lipid mixture in this context applies to the lipid mixture component of both the aqueous dispersion and the nucleic acid-lipid particle.
  • the lipid mixture in the compositions (including the aqueous dispersion, nucleic acid- lipid particles and functionalized nucleic acid-lipid particles) of the present invention may further comprise one or more additional lipids.
  • the one or more additional lipids comprise an anionic amphiphile, as defined and exemplified below.
  • the one or more additional lipids comprise a neutral or zwitterionic lipid, as defined and exemplified below.
  • the one or more additional lipids comprise a steroid, as defined and exemplified below.
  • the one or more additional lipids comprise a neutral lipid, as defined and exemplified below.
  • the one or more additional lipids comprise a neutral lipid (such as a steroid), as defined and exemplified below.
  • compositions (including the aqueous dispersion, nucleic acid-lipid particles and functionalized nucleic acid-lipid particles) of the present invention may also additionally comprise a neutral lipid.
  • the neutral lipid is preferably a neutral phospholipid.
  • the phospholipid may be zwitterionic (i.e. it carries both a positive and a negative charge, so that it is neutral at a pH ranging around neutral).
  • the phospholipid is selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins.
  • the hydrocarbyl portion of the acyl moieties of phospholipids is as defined above, but is preferably an alkyl group (as defined above) having 6 to 40, preferably 8 to 24, carbon atoms or an alkenyl group (as defined above) having 6 to 40, preferably 14 to 22, carbon atoms and 1 to 6 carbon-carbon double bonds.
  • the acyl parts of the phospholipids may be the same or different.
  • the acyl moieties are saturated fatty acid moieties having 8 to 24 carbon atoms (including the acyl carbon), preferably selected from the group consisting of lignoceroyl, behenoyl, arachidoyl, stearoyl, palmitoyl, myristoyl, lauroyl, decanoyl and octanoyl moieties.
  • neutral phospholipids have a T m of 30°C or higher and are selected from di-stearoyl or di-palmitoyl or stearoyl-palmitoyl moieties.
  • the acyl moieties are unsaturated fatty acid moieties having 14 to 22 carbon atoms (including the acyl carbon), preferably selected from the group consisting of oleoyl, linoyl, and lineoyl moieties.
  • diacylphosphatidylcholines such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine (DLPC), dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), l-o
  • the neutral lipid is selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DOPE, and SM, or a mixture of any thereof.
  • compositions described herein comprise a compound of formula (A), as defined herein, a cationic or cationically ionizable lipid (as defined herein) and a phospholipid.
  • the compositions described herein comprise a compound of formula (A), as defined herein, a cationic or cationically ionizable lipid and a phospholipid selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DOPE, and SM, or a mixture of any thereof.
  • the neutral lipid is present in the lipid mixture in an amount of about 1 mol % to about 40 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is present in the lipid mixture in an amount of about 2 mol % to about 25 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is present in the lipid mixture in an amount of from about 5 mol % to about 15 mol % of the total lipids present in the lipid mixture.
  • the neutral lipid is a phospholipid and is present in the lipid mixture in an amount of about 1 mol % to about 40 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is a phospholipid and is present in the lipid mixture in an amount of about 2 mol % to about 25 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is a phospholipid and is present in the lipid mixture in an amount of from about 5 mol % to about 15 mol % of the total lipids present in the lipid mixture.
  • the neutral lipid is a phosphatidylcholine and is present in the lipid mixture in an amount of about 1 mol % to about 40 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is a phosphatidylcholine and is present in the lipid mixture in an amount of about 2 mol % to about 25 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is a phosphatidylcholine and is present in the lipid mixture in an amount of from about 5 mol % to about 15 mol % of the total lipids present in the lipid mixture.
  • the neutral lipid is DSPC and is present in the lipid mixture in an amount of about 1 mol % to about 40 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is DSPC and is present in the lipid mixture in an amount of about 2 mol % to about 25 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is DSPC and is present in the lipid mixture in an amount of from about 5 mol % to about 15 mol % of the total lipids present in the lipid mixture.
  • lipid mixture in this context applies to the lipid mixture component of both the aqueous dispersion and the nucleic acid-lipid particle.
  • compositions (including the aqueous dispersion, nucleic acid-lipid particles and functionalized nucleic acid-lipid particles) of the present invention may also comprise a steroid.
  • the steroid comprises a sterol.
  • the steroid is cholesterol.
  • the lipid nanoparticle compositions described herein comprise a cationically ionizable lipid (as defined herein) and cholesterol.
  • the steroid is present in an amount ranging from about 10 mol % to about 65 mol % of the total lipids present in the lipid mixture. In one embodiment, the steroid is present in an amount ranging from about 20 mol % to about 60 mol % of the total lipids present in the lipid mixture. In one embodiment, the steroid is present in an amount ranging from about 30 mol % to about 50 mol % of the total lipids present in the lipid mixture.
  • the combined concentration of the neutral lipid in particular, one or more phospholipids, in particular a phosphatidylcholine such as DSPC) and steroid (in particular, cholesterol) may comprise from about 0 mol % to about 70 mol %, such as from about 2 mol % to about 60 mol %, from about 5 mol % to about 55 mol %, from about 5 mol % to about 50 mol %, from of the total lipids present in the lipid mixture.
  • the term “lipid mixture” in this context applies to the lipid mixture component of both the aqueous dispersion, the nucleic acid-lipid particle, and the functionalized nucleic acid-lipid particle.
  • compositions described herein may also contain a grafted lipid.
  • grafted lipid in its broadest sense means a lipid or lipid-like material, as defined above (either in a broadest aspect or a preferred aspect) conjugated to a polymer, as defined above (either in a broadest aspect or a preferred aspect”).
  • the grafted lipid is capable of acting as a stealth lipid.
  • stealth lipid means a stealth polymer (as defined above) conjugated to a lipid (as defined herein).
  • stealth polymer means a polymer (as defined above) having the following features: (a) polar (hydrophilic) functional groups; (b) hydrogen bond acceptor groups, (c) no hydrogen bond donor groups; and (d) no net charge.
  • a stealth polymer is designed to sterically stabilize a lipid particle by forming a protective hydrophilic layer that shields the hydrophobic lipid layer.
  • a stealth polymer 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 grafted lipid is a polyethylene-glycol conjugated lipid (also known as a PEG-lipid or PEGylated lipid).
  • PEGylated lipid refers to a molecule comprising both a lipid portion and a polyethylene glycol portion.
  • PEGylated lipids are known in the art.
  • the PEG-lipid may comprise 5-1000, 5-500, 5- 100, 5-50, 8-1000, 8-500, 8-100, 8-50, 10-1000, 10-500, 10-100, or 10-50, ethylene glycol repeating units, which may be consecutive.
  • 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 1 -(monomethoxy -poly ethyleneglycol)- 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- 1 -0-(co-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(te)
  • 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-[(co-methoxy poly(ethylene 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.
  • grafted lipids include poly(sarcosine) (pSar)-conjugated lipids, poly(oxazoline) (POX)-conjugated lipids; poly(oxazine) (POZ)-conjugated lipids, poly(vinyl pyrrolidone) (PVP)-conjugated lipids; poly(A-(2-hydroxypropyl)- methacrylamide) (pHPMA)-conjugated lipids; poly(dehydroalanine) (pDha)- conjugated lipids; poly(aminoethoxy ethoxy acetic acid) (pAEEA)-conjugated lipids and poly(2-methylaminoethoxy ethoxy acetic acid) (pmAEEA)-conjugated lipids.
  • pSar poly(sarcosine)
  • POX poly(oxazoline)
  • POZ poly(oxazine)
  • PVP
  • the grafted lipid is a polysarcosine-conjugated lipid, also referred to herein as sarcosinylated lipid or pSar-lipid.
  • sarcosinylated lipid refers to a molecule comprising both a lipid portion and a polysarcosine (poly(N- methylglycine) portion, the polysarcosine portion having the repeating unit shown below: wherein x refers to the number of sarcosine units.
  • the polysarcosine may comprise from 2 to 200, from 2 to 100, from 5 to 200, from 5 to 100, from 10 to 200, from 10 to 100, optionally from 5 to 80, preferably from 10 to 70 sarcosine units.
  • the grafted lipid is a polyoxazoline (POX)-conjugated and/or a polyoxazine (POZ)-conjugated lipid and/or a POX/POZ-conjugated lipid, also referred to herein as a conjugate of a POX and/or POZ polymer and one or more hydrophobic chains or as oxazolinylated and/or oxazinylated lipid or POX and/or POZ-lipid.
  • POX polyoxazoline
  • POZ polyoxazine
  • oxazolinylated lipid or "POX-lipid” refers to a molecule comprising both a lipid portion and a polyoxazoline portion, the polyoxazoline portion (pOx) having the repeating unit shown below.
  • oxazinylated lipid or “POZ-lipid” refers to a molecule comprising both a lipid portion and a polyoxazine portion, the polyoxazine (pOz) portion having the repeating unit shown below.
  • oxazolinylated/ oxazinylated lipid or "POX/POZ-lipid” or “POXZ-lipid” refers to a molecule comprising both a lipid portion and a portion of a copolymer of polyoxazoline and poly oxazine, i.e. a polymer having both the pOx and pOz repeating units shown below: wherein x refers to the number of pOx and/or pOz units.
  • the total number of pOx and/or pOz repeating units in the polymer may comprise from 2 to 200, from 2 to 100, from 5 to 200, from 5 to 100, from 10 to 200, from 10 to 100, optionally from 5 to 80, preferably from 10 to 70 pOx and/or pOz units.
  • the grafted lipid is a poly(vinyl pyrrolidone) (PVP)-conjugated lipid.
  • the lipid nanoparticle composition is substantially free (as defined above, either in its broadest aspect of a preferred aspect) of a poly(vinyl pyrrolidone) (PVP) conjugated to a lipid.
  • PVP poly(vinyl pyrrolidone)
  • the term “poly(vinyl pyrrolidone)” or “PVP” means a polymer having a vinyl pyrrolidine repeating unit, i.e. the repeating unit shown below.
  • the grafted lipid is a poly(7V-(2-hydroxypropyl)methacrylamide) (pHPMA)-conjugated lipid.
  • the lipid nanoparticle composition is substantially free (as defined above, either in its broadest aspect of a preferred aspect) of polyCV-(2-hydroxypropyl)methacrylamide) (pHPMA) conjugated to a lipid.
  • poly(A-(2-hydroxypropyl)-methacrylamide” or “pHPMA” means a polymer having the repeating unit shown below.
  • the grafted lipid is a poly(dehydroalanine) (pDha)-conjugated lipid.
  • pDha means a polymer having the repeating unit shown below.
  • the grafted lipid is an amphiphilic oligoethylene glycol (OEG)- conjugated lipid.
  • amphiphilic oligoethylene glycol (OEG)-conjugated lipids include poly(aminoethyl-ethylene glycol acetyl) (pAEEA) and/or poly(methylaminoethyl-ethylene glycol acetyl) (pmAEEA).
  • pAEEA and “pmAEAA” means a polymer having the repeating unit shown below: wherein x refers to the total number of pAEEA and/or pmAEEA units in the polymer.
  • the total number of pAEEA and/or pmAEEA repeating units in the polymer may comprise from 1 to 100, from 5 to 50, from 5 to 25, preferably from 7 to 14.
  • the one or more additional lipids comprise a grafted lipid, preferably wherein the grafted lipid is selected from the group consisting of a poly(alkylene glycol)-conjugated lipid, a poly(sarcosinate)-conjugated lipid, a poly(oxazoline) (POX)-conjugated lipid; a poly(oxazine) (POZ)-conjugated lipid; a poly(vinyl pyrrolidone) (PVP)-conjugated lipid; a poly(A-(2-hydroxypropyl)- methacrylamide) (pHPMA)-conjugated lipid; a poly(dehydroalanine) (pDha)- conjugated lipid; a poly(aminoethoxy ethoxy acetic acid) (pAEEA)-conjugated lipid; and a poly(2-methylaminoethoxy ethoxy acetic acid) (pm
  • the grafted lipid is a poly(aminoethoxy ethoxy acetic acid) (pAEEA)-conjugated lipid as defined above, either in its broadest aspect or a preferred aspect.
  • the grafted lipid is present in the lipid mixture in an amount of 0.5 to 10 mol% of the total lipids present in the lipid mixture.
  • the grafted lipid is present in the lipid mixture in an amount of 0.2 to 5 mol% of the total lipids present in the lipid mixture.
  • the grafted lipid is present in the lipid mixture in an amount of 1 to 2.5 mol% of the total lipids present in the lipid mixture.
  • the grafted lipid may comprise a mixture of (i) a grafted lipid selected from the group consisting of pSar-conjugated lipids; POX-conjugated lipids; POZ- conjugated lipids, PVP-conjugated lipids; pHPMA-conjugated lipids; pDha- conjugated lipids; pAEEA-conjugated lipids and pmAEEA-conjugated lipids, and (ii) a peptide conjugated lipid.
  • lipid mixture in this context applies to the lipid mixture component of the aqueous dispersion, the nucleic acid-lipid particle, and the functionalized nucleic acid-lipid particle.
  • the functionalized particles of the present invention comprise an ALFA-tag.
  • an ALFA-tag comprises the amino acid sequence -AA0-AA1- AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12-AA13-AA14-, wherein the amino acids of AAO, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13 and AA14 are: AAO is Pro or deleted;
  • AA1 is Ser, Gly, Thr, or Pro
  • AA2 is Arg, Gly, Ala, Glu, or Pro
  • a A3 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; AA1 1 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 (SEQ ID NO: 75), P SRLEEELRRRLTE (SEQ ID NO: 78), SRLEEELRRRLTEP (SEQ ID NO: 79), and PSRLEEELRRRLTEP (SEQ ID NO: 80).
  • 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 which are not XI and X2 are:
  • AAO is Pro or deleted
  • AA1 is Ser, Gly, Thr, or Pro
  • AA2 is Arg, Gly, Ala, Glu, or Pro
  • a A3 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
  • AA1 1 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 -AA0-AAl-AA2-AA3-AA4-cyclo(Xl-AA6-AA7-AA8-X2)-Arg-Leu-AA12-AA13- AA14-, -AA0-AAl-AA2-AA3-AA4-cyclo(Xl-AA6-AA7-X2)-AA9-Arg-Leu-AA12-AA13- AA14-, -AA0-AAl-AA2-AA3-AA4-AA5-AA6-AA7-AA8-cyclo(Xl-Arg-Leu-AA12-X2)- AA14-, -AA0-AAl-AA2-AA3-AA4-AA5-cyclo(Xl-AA7-AA8-X2)-Arg-Leu-AA12-AA13- AA14-, -AA0-AAl-AA2-AA3-AA4-AA5-cyclo(Xl-AA7-AA8-X2)-Arg-Leu
  • AA1 is Ser, Gly, Thr, or Pro
  • AA2 is Arg, Gly, Ala, Glu, or Pro
  • a A3 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, DGlu, Asp, DAsp, Lys, DLys, hLys, DhLys, Orn, DOm, Dab, DDab, Dap, DDap, Cys, DCys, hCys, DhCys, Pen, and DPen, with the proviso that when XI is Glu, DGlu, Asp, or DAsp, X2 is Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, or DDap; when XI is Lys, DLys, hLys, DhLys, Orn, DOm, Dab, DDab, Dap, or DDap, X2 is Glu, DGlu, Asp, or DAsp; and when XI is Cys,
  • XI is Glu and X2 is Lys.
  • -cyclo(Glu — — Lys)-, -c(Glu - Lys)-, -cyclo 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:
  • cyclized amino acid sequences of the above-identified general formulae include, for example, -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu- (SEQ ID NO:

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Abstract

La présente invention concerne de manière générale des particules fonctionnalisées contenant des fractions capables de se lier à l'immunoglobuline D (IgD), des procédés de production de celles-ci, et des compositions pharmaceutiques les contenant et leurs utilisations en médecine.
PCT/EP2024/080804 2023-10-31 2024-10-31 Particules, compositions et procédés Pending WO2025093665A1 (fr)

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