Particles, compositions, and methods

Abstract

The present invention relates generally to functionalized particles containing moieties capable of binding immunoglobulin D (IgD), methods for making the same, and pharmaceutical compositions containing the same and their use in medicine.

Description

Technical Field

[0001] This disclosure generally relates to functionalized lipid particles containing a portion capable of binding immunoglobulin D (IgD), methods for preparing the same, and pharmaceutical compositions containing the same and their use in medicine.

[0002] In particular, this invention relates to particles comprising single-domain antibodies (sdAbs) targeting IgD. The invention also relates to variable heavy chain domain antibodies (VHHs) comprising sdAbs, conjugates, functionalized lipid particles, chimeric antigen receptors, and immune cell engagers. IgD-targeting methods can be used for payload delivery to B cells and for the treatment of diseases. Background of the Invention

[0004] Immunoglobulin D (IgD) is an antibody isotype co-expressed with IgM on mature B cells as part of the B cell receptor complex along with CD79a and b. IgD can also be detected in circulation in a soluble form, as with other members of the immunoglobulin (Ig) family, although at very low concentrations (approximately 50-fold lower than IgM and less than 0.25% of total serum Ig; see Nguyen et al.; 2022; International Reviews of Immunology; 41(2); 107-122). The function of IgD is not fully understood, and earlier studies have shown that IgD and IgM are functionally interchangeable. IgD knockout has not shown impairment of B cell development; however, other studies have shown that IgD can control primary immune responses and affinity maturation (Amendt et al.; 2021; Front. Immunol.; 12).

[0005] Developing agents that can specifically bind to IgD can be used in a variety of situations; for example, in cases where it is desirable to target B cells to guide treatment and / or diagnosis of the entity.

[0006] Although immunoglobulin D (IgD) was discovered more than 50 years ago, its specific function in the human immune system remains poorly understood compared to other members of the immunoglobulin family. Only a few IgD-specific antibodies have been described in the literature (see Nguyen et al., 2022, International Reviews of Immunology 41(2):1-24, and Nguyen et al., J. Autoimmunity, 2010, 35(1), 86-97).

[0007] Therefore, there is a need for agents that can specifically bind to IgD, and for particles that can carry such agents to deliver the payload to IgD-expressing cells (such as B cells). Invention Overview

[0009] According to a first aspect of the invention, functionalized particles are provided, comprising:

[0010] (a) One or more particulate-forming components;

[0011] (b) payloads (such as nucleic acid payloads); and

[0012] (c) The portion that can bind to immunoglobulin D (IgD).

[0013] In one implementation, the functionalized particles comprise:

[0014] (a) One or more particulate-forming components;

[0015] (b) Loading payloads (such as nucleic acid payloads);

[0016] (c) A linker compound comprising:

[0017] (i) The portion capable of incorporating linker compounds into the particles, and

[0018] (ii) the first interaction part; and

[0019] (d) Docking compounds, which include:

[0020] (i) the second interaction component, and

[0021] (ii) The portion that can bind to IgD;

[0022] The first and second interacting parts can combine with each other.

[0023] In the above implementation scheme, the functionalized particles include:

[0024] (a) One or more particulate-forming components;

[0025] (b) Loading payloads (such as nucleic acid payloads);

[0026] (c) Linker compounds of formula (A):

[0027] L‐X1‐P‐X2‐B1 (A); and

[0028] (d) Docking compounds of formula (I):

[0029] B2-X3-B3 (I)

[0030] in:

[0031] P either does not exist or contains a polymer;

[0032] L contains a portion capable of incorporating the compound into the particles, which is attached to B1 when P is absent, or to the first end of polymer P when P is present.

[0033] B1 contains a portion that can bind to B2. When P is absent, a portion of B1 is attached to L, or when P is present, it is attached to the second end of polymer P.

[0034] X1 and X2 are either independent or connected parts;

[0035] B2 contains a portion that can combine with B1;

[0036] X3 is missing or is a connection part; and

[0037] B3 contains a portion that can bind to IgD.

[0038] In another embodiment, the functionalized particles comprise:

[0039] (a) One or more particulate-forming components;

[0040] (b) payloads, such as nucleic acid payloads; and

[0041] (c) Contains the following compounds:

[0042] (i) The portion capable of incorporating linker compounds into the particles, and

[0043] (ii) The portion that can bind to immunoglobulin D (IgD).

[0044] This implementation scheme is referred to below as the "direct binding implementation scheme" and compound (c) is referred to as the "direct binding compound".

[0045] In this embodiment, compound (c) is preferably a compound of formula (A'):

[0046] L‐X1‐P‐X2‐B' (A')

[0047] in:

[0048] P either does not exist or contains a polymer;

[0049] L contains a portion capable of incorporating the compound into the particles. When P is absent, L is attached to B, or when P is present, it is attached to the first end of polymer P.

[0050] B' contains a portion that can bind to IgD. When P is absent, part of B' is attached to L, or when P is present, it is attached to the second end of polymer P.

[0051] X1 either does not exist or is the first connection part; and

[0052] X2 either does not exist or is a second connection part.

[0053] In this embodiment, B' is preferably selected from the group consisting of a free antibody, an antibody-like molecule, a VHH, or a peptide. In one embodiment, B' is a VHH. In another embodiment, B' is a peptide.

[0054] According to another aspect of the present invention, a method for forming the functionalized particles of the present invention is provided, the method comprising:

[0055] (a) Forming particles containing compounds of formula (A) capable of interacting with compounds of formula (I), and

[0056] (b) Mix the particles formed in step (a) with the compound of formula (I),

[0057] This causes the compound of formula (I) to interact with particles containing the compound of formula (A).

[0058] According to another aspect of the invention, a pharmaceutical composition is provided comprising functionalized lipid particles and a carrier according to the invention.

[0059] According to another aspect of the present invention, a kit is provided comprising:

[0060] (i) Particles containing compounds of formula (A) as defined herein;

[0061] (ii) payloads, such as nucleic acids; and

[0062] (iii) Compounds of formula (I) as defined herein.

[0063] According to another aspect of the invention, an in vitro method is provided, which includes contacting cells with functionalized lipid particles according to the invention.

[0064] According to another aspect of the invention, functionalized lipid particles according to the invention are provided for use in methods of treating or diagnosing diseases.

[0065] According to another aspect of the invention, functionalized lipid particles according to the invention are provided for targeted delivery of payloads (such as nucleic acid payloads) to IgD-positive cells.

[0066] According to another aspect of the invention, functionalized lipid particles according to the invention are provided for targeted delivery of payloads (such as nucleic acid payloads) to B cells.

[0067] In another aspect of the invention, functionalized particles are provided, comprising:

[0068] (a) One or more particulate-forming components;

[0069] (b) Nucleic acid payload;

[0070] (c) A linker compound comprising:

[0071] (i) The portion capable of incorporating linker compounds into the particles, and

[0072] (ii) the first interaction part; and

[0073] (d) Docking compounds, which include:

[0074] (i) the second interaction component, and

[0075] (ii) The portion that can bind to cell surface antigens;

[0076] The first and second interacting parts can combine with each other;

[0077] One of the first and second interacting portions contains an sdAb capable of binding to the ALFA-tagged peptide. According to Kabat's annotation, the sdAb includes a CDR containing the following sequence:

[0078] CDR1-ALNAMAMG (SEQ ID NO: 137);

[0079] CDR2 – AVSERGNTYYRDSVKG (SEQ ID NO: 138); and

[0080] CDR3 – LEDRVDSFHDY (SEQ ID NO: 139).

[0081] Advantages and unexpected discoveries

[0082] The inventors of this invention have unexpectedly determined that functionalized particles (such as lipid particles) containing a targeting portion that binds IgD exhibit advantageous properties when targeting B cells. For example, functionalized lipid particles containing a targeting portion that binds IgD show advantages over particles having a targeting portion that targets other B cell surface molecules (e.g., IgM). In particular, functionalized nucleic acid-lipid particles containing a targeting portion that binds IgD can have increased transfection efficiency compared to particles having a targeting portion that targets IgM.

[0083] Furthermore, the anti-IgD single-domain antibody (sdAb – particularly VHH) molecules presented herein possess numerous advantageous properties, including when incorporated into the functionalized particles of this invention. For example, sdAbs are capable of specifically binding to both human and rhesus monkey (Macaca mulatta) IgD; they are beneficial in preclinical animal models, such as preclinical toxicology assessments. This can also provide advantages when advancing from preclinical to clinical studies for indications such as HIV / AIDS. For instance, the same cross-reactive IgD binder can be used in two studies, instead of using a rhesus monkey-specific IgD binder in preclinical studies and different human-specific IgD binders in clinical studies.

[0084] Compared to other immunoglobulins, the sdAb of the present invention may also have selective binding to IgD. Suitably, the sdAb of the present invention can bind IgD but does not bind or is substantially unable to bind one or more other immunoglobulins. For example, the sdAb of the present invention may not bind or is substantially unable to bind IgA, IgE, IgG, and / or IgM. Suitably, the sdAb of the present invention is internalized into the cell after binding to IgD on the cell surface; for example, this can be advantageous for the delivery of the payload to the target cell. Suitably, after the sdAb of the present invention binds to IgD on the surface of B cells, it can be able to activate B cells. Suitably, the sdAb of the present invention can retain its ability to bind IgD, particularly IgD expressed on the cell surface, in the presence of serum (e.g., human serum). Brief description of the attached diagram

[0086] Figure 1 - (A) Immunization Schedule. Two alpacas were immunized three times (days 0, 26, and 67) with mRNA LNP (500 µg human IgD Fc-CP) and twice (days 84 and 111) with 100 µg rhesus monkey IgD Fc + 100 µg rhesus monkey IgD Fc-CP. Blood was drawn from the animals on days 74 and 117 for titer analysis, cDNA preparation, and screening (day 117 only). Serum from immunized animals was diluted 1:100–1:100000 in assay buffer and tested for binding to (B) immobilized human IgD Fc-CP protein or (C) rhesus monkey IgD Fc-CP protein via ELISA. CP = linker peptide.

[0087] Figure 2- Binding properties of 27 anti-IgD VHH candidates derived from screening and HIT ELISA. The kinetic binding behavior of anti-IgD candidates against immobilized IgD Fc moieties from human or rhesus monkeys, containing or lacking a linker peptide region (CP), was determined using biolayer interferometry (BLI). Immobilization was achieved via a terminal His-tag on the IgD Fc domain until a response of 0.7 nm was achieved. VHH-GFP fusion was allowed to associate for 600 seconds, followed by dissociation for 600 seconds. Serial dilutions of VHH were used, starting at 50 or 200 nM. Kd, Ka (association rate), and Kdis (dissociation rate) values ​​for each candidate are given. Cellular binding and specificity of IgD VHH were analyzed by incubating 500 and 1000 nM IgD VHH clones with isolated B cells at 4°C for 30 min. After incubation, cells were washed and stained with appropriate antibodies. Cells were then fixed and analyzed via FACs. Nb: Unbound.

[0088] Figure 3 – Specificity analysis of selected IgD-Fc binders in VHH-GFP form. Microtiter plates were coated with 5 pmol of human or rhesus monkey IgD-Fc-CP protein, human IgA, human IgE, human IgG, or human IgM. Wells were then incubated with 1000, 100, and 10 nM VHH in the 3xFLAG-GFP fusion form, diluted only in assay buffer or pre-blocked with 50% human serum. Anti-IgD VHH, specific only for human IgD, served as a positive control. No VHH was added as a negative control. Binding to different coated antigens was detected using appropriate secondary antibodies.

[0089] Figure 4 - An overview of the biophysical properties and binding kinetics of 14 selected IgD binders in NbALFA fusion form. Purity was analyzed by SEC and values ​​indicated the percentage of area under the curve (AUC) of the main peak. Monodispersity (cumulative radius (in nm) and PDI) was analyzed using DLS with a Prometheus Panta device. Stability was measured by Nano-DSF on a Prometheus Panta device. The binding kinetics of the anti-IgD candidates to human and rhesus monkey IgD-Fc proteins were determined by BLI. For this purpose, NbALFA fusions were immobilized on the tip of a SAX2.0 sensor via a biotinylated ALFA-tagged peptide for capture. After a 60-second baseline in KB, association was measured for 600 seconds using seven different concentrations of the corresponding target IgD-Fc protein, starting at 50 nM and serially diluted 1:1 to 0.78125 nM. As a reference, KB buffer without IgD-Fc was measured. Dissociation was obtained after 600 seconds in KB buffer.

[0090] Figure 5 - Cell binding and internalization: Cells were incubated with various VHH clones at 4°C (for surface binding) and 37°C (for internalization). VHH clones were detected via FLAG tags and analyzed by FACs.

[0091] Figure 6 - PBMCs treated with aIgD LNP. PBMCs were treated with 500 ng of aIgD LNP encapsulating Thy1.1 RNA. (A) Examples of FACs analysis for identifying Thy1.1 expression in different cell types by using CD3 to identify T cells, CD19 to identify B cells, and CD14 to identify monocytes. (B) Graphical representation of Thy1.1 expression in different clones and cell types.

[0092] Figure 7 - Isolated B cells and PBMCs treated with 1000 ng of aIgDLNP containing Thy1.1 RNA and luciferase in the presence of 50% human serum. (A) Thy1.1 RNA expression in isolated B cells treated with various aIgD LNPs was analyzed by FACS. The graph was gated for CD20 and Thy1.1 double-positive cells after treatment with or without human serum. (B) Thy1.1 RNA expression in PBMCs treated with various aIgD LNPs was analyzed by FACS. T, B, and monocytes were identified by expression of CD3, CD19, and CD14, respectively. Transfection was detected by Thy1.1 expression in each subset. (C) Graphical representation of B cell transfection in PBMCs treated with and without serum (n=3). (D) Thy1.1 RNA expression in PBMCs treated with control nonfunctionalized LNPs and aIgM LNPs in or without human serum was analyzed by FACS. T cells, B cells, and monocytes are identified by the expression of CD3, CD19, and CD14, respectively. Transfection was detected by the expression of Thy1.1 in each subset.

[0093] Figure 8 – Table 1 – Summary of VHH CDRs defined by the Chothia-Nanobody, IMGT, and Kabat systems

[0094] Figure 9 –Table 2- Summary of VHH

[0095] Figure 10 –Table 3– Summary of VHH-NbALFA fusions

[0096] Figure 11- Isolated human B cells were treated with control nonfunctionalized LNPs and various aIgDLNPs for 30 minutes in the presence of 50% human serum. After particle treatment, the cells were incubated overnight and their activation status was analyzed by the expression of the activation marker CD86.

[0097] Figure 12 - Physicochemical characterization of LNPs via dynamic light scattering (DLS): Wyatt DynaPro PlateReader-II (single-angle λ = 830 nm (75 mW), acquisition time: 10 s and cumulative acquisition: 10x). The device was used to measure the size and polydispersity index (PDI) of LNPs functionalized with 14 aIgD NbALFA ligand variants (N / P = 4, DODMA / Chol / DOPE / C16-PEG-ceramide / DSPE-PEG2k-ALFA 40 / 48 / 10 / 1.8 / 0.2), where X:L was 4.4, 7, and 14; and the size and polydispersity index of unfunctionalized LNPs with and without ALFA lipids and aIgM LNPs functionalized with aIgM NbALFA ligands as controls. All 45 LNPs tested (with a final concentration of 0.1 mg / mL for the cargo Th1.1 / Luc mRNA_1:1 and a DLS-measured concentration of 0.05 mg / mL, pre-diluted in water by a factor of 20x) showed optimal size: 100–135 d / nm, with PDI < 0.35.

[0098] Figure 13 – Physicochemical characterization of LNPs: Agarose gel electrophoresis (AGE) showed that no detectable free load Thy1.1 and Luc mRNA were present in any formulation. Upper AGE images: Wells 1-2 and 25-26 show negative control LNPs: with and without ALFA lipids; and wells 3 and 27 are positive control functionalized aIgM LNPs (0.2 mol%); wells 4-24 and 28-48 are LNPs functionalized with 14 aIgD NbALFA ligand variants (N / P=4, DODMA / Chol / DOPE / C16-PEG-ceramide / DSPE-PEG2k-Alfa 40 / 48 / 10 / 1.8 / 0.2), where X:L is 4.4, 7, and 14. The final load concentration for all tested LNP formulations was 0.1 mg / mL. A mixture of two free loads (Thy1.1 / Luc mRNA, 1:1 by weight) in amounts of 0.1 µg and 0.5 µg was used as a control sample.

[0099] Figure 14- Physicochemical characterization of LNP: A) Explanation of the functionalization process after aIgD VHH-NbALFA ligand. A) The NbALFA fusion protein was immobilized onto the surface of ALFA LNPs via a high-affinity interaction of NbALFA:ALFA; B) The following were determined by agarose gel electrophoresis: 1) ALFA-free control LNPs (wells 1-3: 1) ALFA-free LNPs; 2) LNPs with 0.2 mol% ALFA lipids; 3) aIgM LNPs; LNPs functionalized with the first 6 selected aIgD variants (pNT3129, pNT3133, pNT3193, pNT3194, pNT3197, pNT3198) (wells 4-9, N / P=4, DODMA / Chol / DOPE / C16-PEG-ceramide / DSPE-PEG2k-Alfa 40 / 48 / 10 / 1.8 / 0.2, where X:L is 4.4, and the final loading concentration is 0.1 mg / mL); C) Zeta potential measurements: 1) LNPs without ALFA lipids, 2) LNPs with 0.2 mol% ALFA lipids, 3) aIgM LNPs, and 4) LNPs functionalized with 6 aIgD ligand variants. LNPs were pre-diluted at a factor of 50x with 20 mM HEPES buffer.

[0100] Figure 15 – Isolated B cells stained with the proliferation dye CTV were treated with ALFA (unfunctionalized) or functionalized aIgDpNT3194 or pNT3198 LNP. Cells were incubated for 4 days, and their activation status was then analyzed by FACs and proliferation was analyzed by loss of CTV dye.

[0101] Figure 16 Freshly prepared human PBMCs (A), cynomolgus monkey PBMCs (B), and rhesus monkey PBMCs (C) were incubated with 1000 nM aIgD VHH at 4°C for 30 min, followed by washing with 2X PBS. Cells were then stained with CD20 antibody to differentiate B cell populations and with commercially available human and monkey aIgD antibodies. VHH was stained with a secondary antibody targeting nbALFA. The binding of VHH to the commercially available antibody was then analyzed via FACs.

[0102] Figure 17- Comparison of aIgD VHH with commercially available antibodies. Kinetic binding curves of two different exemplary VHH-NbALFA fusions (A and B), a commercially available anti-monkey IgD antibody (C, ABclonal #A23494), and an anti-human IgD antibody (D, BioLegend #348202) against human (left) and rhesus monkey (right) IgD-Fc antigens. Binding was measured via biolayer interferometry on a Sartorius Octet HTX device. Association and dissociation curves are plotted as solid lines, with the fitted results shown as dashed lines. They are separated by dashed vertical lines. The concentration applied during each association step is shown on the right side of each sensing plot with a significant response. Binding was not observed with the ABclonal antibody in both antigens and with the BioLegend antibody in the rhesus monkey IgD-Fc antigen.

[0103] Figure 18 - DNA delivery to primary human B cells. B cells treated with an LNP formulation encapsulating Sleeping Beauty transposons and the reporter gene Venus were analyzed by FACs over a 10-day period to confirm the delivery and insertion of Venus DNA.

[0104] Detailed Explanation

[0105] The elements of this disclosure will be described in more detail below. These elements are listed together with specific embodiments; however, it should be understood that they can be combined in any way and in any number to create other embodiments. The various examples and preferred embodiments described should not be construed as limiting this disclosure to the explicitly described embodiments. This description should be understood to support and cover embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, unless the context otherwise requires, any permutation and combination of all elements described in this application should be considered as disclosed by the description of this application.

[0106] Preferably, the terms used herein are as defined in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", HGW Leuenberger, B. Nagel, and H. Kölbl, eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995). Unless otherwise stated, practice of this disclosure will employ conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as explained in the literature in the art (see, for example, Organicum, Deutscher Verlag der Wissenschaften, Berlin 1990; Streitwieser / Heathcook, "Organische Chemie", VCH, 1990; Beyer / Walter, "Lehrbuch der Organischen Chemie", S. Hirzel Verlag Stuttgart, 1988; Carey / Sundberg, "Organische Chemie", VCH, 1995; March, "Advanced Organic Chemistry", John Wiley & Sons, 1985; Römpp Chemie Lexikon, Falbe / Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A 30 Laboratory). Manual, 2nd edition, edited by J. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989.

[0107] All methods described herein may be performed in any suitable order unless otherwise stated herein or otherwise obviously contradicted by the context. The use of any and all instances or exemplary language (e.g., “such as”) provided herein is intended only to better illustrate this disclosure and does not limit the scope of this disclosure as otherwise claimed. The language in the specification should not be construed as indicating that any non-claimed element is necessary for the implementation of this disclosure.

[0108] The enumeration of ranges of values ​​in this document is intended only as a shorthand way to refer to each individual value falling within that range. Unless otherwise stated herein, each individual value is incorporated into the specification as if it were enumerated separately herein.

[0109] Throughout this specification, numerous references are cited. Each of these references (including all patents, patent applications, scientific publications, manufacturers' specifications, instructions, etc.), whether mentioned above or below, is hereby incorporated in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to claim prior invention rights over such disclosure.

[0110] definition

[0111] The following provides definitions applicable to all aspects of this disclosure. Unless otherwise stated, the following terms have the following meanings. Any undefined term has its meaning as generally accepted in the field.

[0112] Throughout this specification and the appended claims, unless the context otherwise requires, the word “comprise” and variations such as “comprises” and “comprising” shall be understood to imply inclusion of the stated member, integer, or step, or a group of members, integers, or steps, but not to exclude any other member, integer, or step, or a group of members, integers, or steps. The term “substantially constitutes…” means to exclude any other member, integer, or step of significant importance. The term “comprise” encompasses the term “substantially constitutes…”, which in turn encompasses the term “consistent with…”. Therefore, whenever the term “comprises” appears in this application, it may be replaced by the terms “substantially constitutes…” or “consistent with…”. Similarly, whenever the term “substantially constitutes…” appears in this application, it may be replaced by the terms “consistent with…”.

[0113] Unless otherwise stated herein or explicitly contradicted by the context, the terms “a,” “an,” and “the,” as well as similar references, used in the context of describing this disclosure (especially in the context of the claims) should be interpreted to cover both the singular and the plural.

[0114] As used herein, “and / or” should be considered as a specific disclosure of each of the two specified features or components with or without the other. For example, “X and / or Y” should be considered as a specific disclosure of each of (i) X, (ii) Y, and (iii) X and Y, as if each were stated separately herein.

[0115] In the context of this disclosure, the term "about" refers to a range of precision that a person skilled in the art would understand to still ensure the technical effect of the feature in question. This term typically indicates a deviation from the indicated numerical value of ±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%. For example, with respect to pH values, the term "about" may, in a preferred case, indicate a deviation from the indicated numerical value of up to 0.3. As a person skilled in the art will understand, the specific deviation of such a numerical value for a given technical effect will depend on the nature of the technical effect. For example, natural or biotechnological effects may generally have a larger such deviation than those of artificial or engineered effects.

[0116] As used herein, the expression "substantially free of X" means that the compositions described herein are free of X in such a practically practicable manner. For example, if a mixture is substantially free of X, the amount of X in the mixture may be less than 1% by weight based on the total weight of the mixture (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). This document defines the specific meaning of the term "substantially free of X" in relation to certain components of the composition.

[0117] As used herein, “physiological pH” refers to a pH of about 7.5 or about 7.4. In some embodiments, the physiological pH is 7.3 to 7.5. In some embodiments, the physiological pH is 7.35 to 7.45. In some embodiments, the physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.

[0118] As used herein, “physiological conditions” refers to conditions (particularly pH and temperature) in a living subject, particularly in a human. Preferably, physiological conditions mean physiological pH and / or a temperature of approximately 37°C.

[0119] As used in this disclosure, "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.

[0120] As used in this disclosure, "mol% of 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. In this context, in some embodiments, the terms "total lipids" and / or "total lipid mixture" include lipids and lipid-like materials.

[0121] As used herein, the term "hydrocarbon group" refers to a monovalent organic group obtained by removing a hydrogen atom from a hydrocarbon molecule. In some embodiments, the hydrocarbon group is acyclic, for example, linear or branched. Typical examples of hydrocarbon groups include alkyl, alkenyl, alkynyl, cycloalkyl, aryl groups, and combinations thereof (such as arylalkyl (aralkyl) etc.). A specific example of a hydrocarbon group is a C 1-40 Alkyl groups (such as C) 6-40 Alkyl, C 6-30 Alkyl, C 6-20 Alkyl, or C 10-20 Alkyl groups, having 1, 2, or 3 double bonds (C). 2-40 alkenyl (such as C) 6-40 alkenyl, C 6-30 alkenyl, or C 6-20 alkenyl), aryl and aryl (C 1-6 Alkyl group). In some embodiments, the hydrocarbon group is optionally substituted by one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituent selected from List A.

[0122] The term "heteroalkyl group" means a hydrocarbon group as defined above, wherein 1, 2, 3, or 4 carbon atoms in the hydrocarbon group are replaced by heteroatoms of oxygen, nitrogen, silicon, selenium, phosphorus, or sulfur, preferably O, S, or N. In one embodiment, the heteroalkyl group is substituted by one or more, such as 1, 2, or 3, such as 1 or 2, such as 1 selected from List A.

[0123] The term "aliphatic" refers to a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more unsaturated units, or a monocyclic or bicyclic hydrocarbon (also referred to herein as "cycloaliphatic") that is fully saturated or contains one or more unsaturated units but is not aromatic, having a single attachment site or more than one attachment site with the rest of the molecule. Unless otherwise stated, the aliphatic group contains 1-12 aliphatic carbon atoms. In some embodiments, the aliphatic group contains 1-6 aliphatic carbon atoms (e.g., C64 ... 1-6 In some embodiments, the aliphatic group contains 1-5 aliphatic carbon atoms (e.g., C10, C20, C30, C40, C50, C60, C70, C80, C9 ... 1-5 In other embodiments, the aliphatic group contains 1-4 aliphatic carbon atoms (e.g., C14, C24, C34, C44, C54, C64, C7 ... 1-4In other embodiments, the aliphatic group contains 1-3 aliphatic carbon atoms (e.g., C10, C20, C30, C40, C50, C60, C70, C80, C9 ... 1-3 In other embodiments, the aliphatic group contains 1-2 aliphatic carbon atoms (e.g., C10, C20, C30, C40, C50, C60, C70, C80, C9 ... 1-2 Suitable aliphatic groups include, but are not limited to, straight-chain or branched, substituted or unsubstituted alkyl, alkenyl, or alkynyl groups and their hybrids. Preferred aliphatic groups are C14 and C24. 1-6 alkyl.

[0124] The term "alkyl" refers to a monovalent group of a saturated straight-chain or branched hydrocarbon. Preferably, the alkyl group comprises 1 to 40 carbon atoms, 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 alkyl groups include methyl, ethyl, propyl, isopropyl (also known as 2-propyl or 1-methylethyl), butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, 1,2-dimethylpropyl, isopentyl, n-hexyl, isohexyl, sec-hexyl, n-heptyl, isoheptyl, n-octyl, 2-ethylhexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecanyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecanyl, n-eicosyl, n-triacyl, n-tetradecyl, etc. "Substituted alkyl" means that one or more hydrogen atoms of an alkyl group (such as one to a maximum number of hydrogen atoms bonded to the alkyl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are replaced by substituents other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). In one embodiment, the alkyl group is replaced by one or more, such as 1, 2, or 3, such as 1 or 2, such as 1 substituent selected from List A. Examples of substituted alkyl groups include chloromethyl, dichloromethyl, fluoromethyl, and difluoromethyl.

[0125] The term "alkylene" refers to a divalent group of a saturated straight-chain or branched hydrocarbon. Preferably, the alkylene group comprises 1 to 40 carbon atoms, 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), butylene isomers (e.g., 1,1-butylene, 1,2-butylene, 2,2-butylene, 1,3-butylene, 2,3-butylene (cis or trans or mixtures thereof), 1,4-butylene, 1,1-isobutylene, 1,2-isobutylene and 1,3-isobutylene), and pentylene isomers (e.g., 1,1-pentylene, 1,2-pentylene, 1,3-pentylene, 1,4-pentylene, 1,5-pentylene, 1,1... -Isopentyl, 1,1-sec-pentyl, 1,1-neopentyl), hexane isomers (e.g., 1,1-hexane, 1,2-hexane, 1,3-hexane, 1,4-hexane, 1,5-hexane, 1,6-hexane and 1,1-isohexane), heptadecyl isomers (e.g., 1,1-heptadecyl, 1,2-heptadecyl, 1,3-heptadecyl, 1,4-hexane, 1,5-hexane, 1,6-hexane and 1,1-isohexane), and heptadecyl isomers (e.g., 1,1-heptadecyl, 1,2-heptadecyl, 1,3-heptadecyl, 1,4-hexane, 1,5-hexane, 1, Examples of alkylene derivatives include 4-heptene, 1,5-heptene, 1,6-heptene, 1,7-heptene, and 1,1-isoheptene, as well as octene isomers (e.g., 1,1-octene, 1,2-octene, 1,3-octene, 1,4-octene, 1,5-octene, 1,6-octene, 1,7-octene, 1,8-octene, and 1,1-isooctene). A straight-chain alkylene moiety having at least three carbon atoms and a free valence at each end can also be referred to as a multiple of the methylene group (e.g., 1,4-butylene can also be referred to as tetramethylene). Often, instead of using the suffix "ylene" for the alkylene moiety specified above, the suffix "diyl" can also be used (e.g., 1,2-butylene can also be referred to as butane-1,2-diyl)."Substituted alkylene" means that one or more hydrogen atoms of the alkylene group (such as one to a maximum number of hydrogen atoms bonded to the alkylene group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are replaced by substituents other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). In one embodiment, the alkylene group is replaced by one or more, such as 1, 2, or 3, such as 1 or 2, such as 1 substituent selected from List A.

[0126] The term "alkenyl" refers to a monovalent group of an unsaturated straight-chain or branched hydrocarbon having at least one carbon-carbon double bond. Typically, the maximum number of carbon-carbon double bonds in an alkenyl group can be an integer, 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 odd, the result of the division is rounded down to the nearest integer. For example, for an alkenyl group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 1 to 6 (e.g., 1 to 4), that is, 1, 2, 3, 4, 5, or 6 carbon-carbon double bonds. Preferably, the alkenyl group contains 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. Therefore, in a preferred embodiment, the alkenyl group comprises 2 to 40 (e.g., 2 to 30, 2 to 20, 2 to 12, 2 to 10) carbon atoms and 1, 2, 3, 4, 5, or 6 (e.g., 1, 2, 3, 4, or 5) carbon-carbon double bonds, such as comprising 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. One or more carbon-carbon double bonds may be in a cis-( Z ) or trans ( EConfiguration. Exemplary alkenyl groups include vinyl, 1-propenyl, 2-propenyl (i.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-Nonenyl, 8-Nonenyl, 1-Dectenyl, 2-Dectenyl, 3-Dectenyl, 4-Dectenyl, 5-Dectenyl, 6-Dectenyl, 7-Dectenyl, 8-Dectenyl, 9-Dectenyl, 1-Undecenyl, 2-Undecenyl, 3-Undecenyl, 4-Undecenyl, 5 5-Undecenyl, 6-Undecenyl, 7-Undecenyl, 8-Undecenyl, 9-Undecenyl, 10-Undecenyl, 1-Dodecenyl, 2-Dodecenyl, 3-Dodecenyl, 4-Dodecenyl, 5-Dodecenyl, 6-Dodecenyl, 7-Dodecenyl, 8-Dodecenyl, 9-Dodecenyl, 10-Dodecenyl, 11-Dodecenyl, etc. "Substituted alkenyl" means that one or more hydrogen atoms of the alkenyl group (such as one to a maximum number of hydrogen atoms bonded to the alkenyl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are replaced by substituents other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). In one embodiment, the alkenyl group is substituted by one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituent selected from List A.

[0127] The term "alkenyl" refers to a divalent group of an unsaturated straight-chain or branched hydrocarbon having at least one carbon-carbon double bond. Typically, the maximum number of carbon-carbon double bonds in an alkenyl group can be an integer, 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 odd, the result of the division is rounded down to the nearest integer. For example, for an alkenyl group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 1 to 6 (e.g., 1 to 4), that is, 1, 2, 3, 4, 5, or 6 carbon-carbon double bonds. Preferably, the alkenyl group comprises 2 to 12 (e.g., 2 to 10) carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (e.g., 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. Therefore, in a preferred embodiment, the alkenyl group comprises 2 to 12 (e.g., 2 to 10) carbon atoms and 1, 2, 3, 4, 5, or 6 (e.g., 1, 2, 3, 4, or 5) carbon-carbon double bonds, more preferably 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. One or more carbon-carbon double bonds may be in a cis-( Z ) or trans ( E Configuration. Exemplary alkenyl groups include ethylene-1,2-diyl, vinylidene (also known as ethenylidene), 1-propen-1,2-diyl, 1-propen-1,3-diyl, 1-propen-2,3-diyl, allyl, 1-butene-1,2-diyl, 1-butene-1,3-diyl, 1-butene-1,4-diyl, 1-butene-2,3-diyl, 1-butene-2,4-diyl, 1-butene-3,4-diyl, 2-butene-1,2-diyl, 2-butene-1,3-diyl, 2-butene-1,4-diyl, 2-butene-2,3-diyl, 2-butene-2,4-diyl, 2-butene-3,4-diyl, etc. "Substituted alkenyl group" means that one or more hydrogen atoms of the alkenyl group (such as one to a maximum number of hydrogen atoms bonded to the alkenyl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are replaced by substituents other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). In one embodiment, the alkenyl group is replaced by one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituent selected from List A.

[0128] The term "alkynyl" refers to a straight-chain or branched monovalent hydrocarbon moiety having at least one carbon-carbon triple bond, wherein the total number of carbon atoms can be six to forty, such as six to thirty, typically six to twenty, such as six to eighteen. The alkynyl group may optionally have one or more carbon-carbon triple bonds. Generally, the maximum number of carbon-carbon triple bonds in an alkynyl group can be an integer, 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 odd, the result of the division is rounded down to the nearest integer. For example, for an alkynyl group having 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, 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. "Substituted alkynyl" means that one or more hydrogen atoms of the alkynyl group (such as one to a maximum number of hydrogen atoms bonded to the alkynyl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are replaced by substituents other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). In one embodiment, the alkynyl group is replaced by one or more, such as 1, 2, or 3, such as 1 or 2, such as 1 selected from List A.

[0129] The term "ynynyl" refers to a divalent group of an unsaturated straight-chain or branched hydrocarbon having at least one carbon-carbon triple bond. Preferably, the ynynyl group has 1 to 6 (e.g., 1 to 4), that is, 1, 2, 3, 4, 5, or 6 carbon-carbon triple bonds. Preferably, the ynynyl group comprises 2 to 12 (e.g., 2 to 10) carbon atoms, that is, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (e.g., 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. Therefore, in a preferred embodiment, the alkenyl group comprises 2 to 12 (e.g., 2 to 10 carbon) atoms and 1, 2, 3, 4, 5, or 6 (e.g., 1, 2, 3, 4, or 5) carbon-carbon triple bonds, more preferably comprising 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon triple bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon triple bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon triple bonds. Exemplary alkenyl groups include acetylene-1,2-diyl, 1-propyne-1,2-diyl, and 1-propyne-1,3-diyl. In one embodiment, the alkenyl group is substituted by one or more, such as 1, 2, or 3, such as 1 or 2, such as 1 substituent selected from List A.

[0130] The terms "cycloalkyl" and "cycloalkenyl" refer to cyclic non-aromatic forms of "alkyl" and "alkenyl" having preferably 3 to 40, such as 3 to 30, such as 3 to 20, such as 3 to 14, such as 3 to 12 or 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms (such as 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, 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 can consist of one ring (monocyclic), two rings (bicyclic), or more than two rings (polycyclic). "Substituted cycloalkyl" means that one or more hydrogen atoms of the cycloalkyl group (such as one to a maximum number of hydrogen atoms bonded to the cycloalkyl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are replaced by substituents other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). In one embodiment, the cycloalkyl or cycloalkenyl group is replaced by one or more, such as 1, 2, or 3, such as 1 or 2, such as 1 selected from List A.

[0131] The terms "cycloalkylene" and "cycloalkenylene" refer to cyclic non-aromatic forms having preferably 3 to 40, such as 3 to 30, such as 3 to 20, such as 3 to 14, such as 3 to 12, or 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms (such as 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary cycloalkylene groups include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and cycloheptylene. Exemplary cycloalkenylene groups include cyclopentenylene and cyclohexenylene.

[0132] The term "aryl" refers to a monovalent group in an aromatic cyclic hydrocarbon. Preferably, 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 may be arranged in a single ring (e.g., phenyl) or two or more fused rings (e.g., naphthyl). Exemplary aryl groups include cyclopropenylonyl, cyclopentadienyl, phenyl, indenyl, naphthyl, azulel, fluorenyl, anthraceneyl, and phenanthryl. Preferably, "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. Aryl does not cover fullerenes. "Substituted aryl" means that one or more hydrogen atoms of an aryl group (such as one to a maximum number of hydrogen atoms bonded to the aryl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 5 or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are replaced by substituents other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). In one embodiment, the aryl group is replaced by one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituent selected from List A. Examples of substituted aryl groups include biphenyl, 2-fluorophenyl, 2-chloro-6-methylphenyl, aniline, 4-hydroxyphenyl, and methoxyphenyl (i.e., 2-, 3-, or 4-methoxyphenyl).

[0133] As used herein, the term "heteroaliphatic" or "heteroaliphatic group" refers to an optionally substituted hydrocarbon moiety having one to five heteroatoms in addition to carbon atoms. It can be straight-chain (i.e., unbranched), branched, or cyclic ("heterocyclic") and can be fully saturated or contain one or more unsaturated units, but it is not aromatic. The term "heteroatom" means nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur and any quaternized form of basic nitrogen. The term "nitrogen" also includes substituted nitrogen. Unless otherwise stated, a heteroaliphatic group contains 1–10 carbon atoms, wherein 1–3 carbon atoms are optionally and independently replaced by heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, the heteroaliphatic group contains 1–4 carbon atoms, wherein 1–2 carbon atoms are optionally and independently replaced by heteroatoms selected from oxygen, nitrogen, and sulfur. In other embodiments, the heteroaliphatic group contains 1–3 carbon atoms, wherein one carbon atom is optionally and independently replaced by a heteroatom selected from oxygen, nitrogen, and sulfur. Suitable heteroaliphatic groups include, but are not limited to, straight-chain or branched heteroalkyl, heteroalkenyl, and heteroynyl groups. For example, heteroaliphatic groups of 1 to 10 atoms include exemplary groups such as -O-CH3, -CH2-O-CH3, -O-CH2-CH2-O-CH2-CH2-O-CH3, etc.

[0134] The term "heteroaryl" or "heteroaromatic ring" refers to an aryl group as defined above, wherein one or more carbon atoms in the aryl group are replaced by heteroatoms of O, S, or N. Preferably, a 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. Alternatively, it refers to an aromatic bicyclic or tricyclic ring system, wherein 1, 2, 3, 4, or 5 carbon atoms are replaced by the same or different heteroatoms of O, N, or S. Preferably, in each ring of the heteroaryl group, the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. Exemplary heteroaryl groups include furanyl, thiophene, oxazolyl, isoxazolyl, oxadiazolyl, pyrroleyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, benzofuranyl, indolyl, isoyindolyl, benzothiophene, 1H-indolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, benzoisoxazolyl, benzothiazolyl, and benzoisothiazolyl. The following groups are listed: benzotriazolyl, quinolinyl, isoquinolinyl, benzodiazinyl, quinoxolinyl, quinazolinyl, benzotriazinyl, pyridazinyl, phenoxazinyl, thiazopyridyl, pyrrolothiazolyl, phenothiazinyl, isobenzofuranyl, chromenyl, xanthonyl, pyrrolizinyl, indazinyl, indazolyl, purinyl, quinazinyl, phthalazinyl, naphthidyl, cinnolinyl, pteridinyl, carbazolyl, phenanthidyl, acridineyl, phenidyl, phenanthrolinyl, and phenazinyl. Exemplary 5- or 6-membered heteroaryl groups include furanyl, thiophene, oxazolyl, isoxazolyl, oxadiazolyl, pyrroleyl, imidazolyl (e.g., 2-imidazolyl), pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridinyl (e.g., 4-pyridinyl), pyrimidinyl, pyrazinyl, triazinyl, and pyridazinyl. "Substituted heteroaryl" means that one or more hydrogen atoms of the heteroaryl group (such as one to a maximum number of hydrogen atoms bonded to the heteroaryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are replaced by substituents other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). In one embodiment, the heteroaryl group is replaced by one or more, such as 1, 2, or 3, such as 1 or 2, such as 1 selected from List A.

[0135] The term "heterocyclic group" or "heterocycle" means a cycloalkyl group as defined above, wherein 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. The heterocyclic group preferably has 1 or 2 rings containing 3 to 10 ring atoms, such as 3, 4, 5, 6, or 7 ring atoms. Preferably, in each ring of the heterocyclic group, the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. The term "heterocyclic group" is also intended to cover partially or completely hydrogenated forms (such as dihydrogen, tetrahydrogen, or perhydrogen forms) of the aforementioned heteroaryl groups. Exemplary heterocyclic groups include morpholino, pyrrolyl, imidazoalkyl, pyrazolyl, piperidinyl (also known as piperidinyl), piperazinyl, dihydrofuranyl and tetrahydrofuranyl, dihydrothiophene and tetrahydrothiophene, dihydropyranyl and tetrahydropyranyl, hexamethylenetetramine (urotropinyl), lactone, lactam, cyclic imide, and cyclic anhydride. "Substituted heterocyclic group" means that one or more hydrogen atoms of the heterocyclic group (such as one to a maximum number of hydrogen atoms bonded to the heterocyclic group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are replaced by substituents other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). In one embodiment, the heterocyclic group is replaced by one or more, such as 1, 2, or 3, such as 1 or 2, such as 1 selected from List A.

[0136] The term "alkylcycloalkyl" means a cycloalkyl group as defined above, which is substituted with an alkyl group as defined above, the cycloalkyl moiety being attached to the remainder of the molecule. Each of the cycloalkyl and alkyl moieties of the group may take any of the broadest or preferred meanings described above. "Substituted alkylcycloalkyl" means that one or more hydrogen atoms of the alkyl or cycloalkyl moiety of the group (such as one to a maximum number of hydrogen atoms bonded to the alkylcycloalkyl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the alkylcycloalkyl group is substituted by one or more, such as 1, 2, or 3, such as 1 or 2, such as 1 selected from List A.

[0137] The term "cycloalkylalkyl" means an alkyl group as defined above, which is substituted with a cycloalkyl group as defined above, the alkyl moiety being attached to the remainder of the molecule. Each of the cycloalkyl and alkyl moieties of the group may take any of the broadest or preferred meanings described above. "Substituted cycloalkylalkyl" means that one or more hydrogen atoms of the alkyl or cycloalkyl moiety of the group (such as one to a maximum number of hydrogen atoms bonded to the cycloalkylalkyl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the cycloalkylalkyl group is substituted by one or more, such as 1, 2, or 3, such as 1 or 2, such as 1 selected from List A.

[0138] The term "alkylcycloalkylalkyl" means an alkyl group as defined above, which is substituted with a cycloalkyl group as defined above, wherein the alkyl moiety is attached to the remainder of the molecule and the cycloalkyl moiety is substituted with another alkyl group. Each of the cycloalkyl and alkyl moieties of the group may take any of the broadest or preferred meanings described above. "Substituted alkylcycloalkylalkyl" means that one or more hydrogen atoms of the alkyl or cycloalkyl moiety of the group (such as one to a maximum number of hydrogen atoms bonded to the alkylcycloalkylalkyl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the alkylcycloalkylalkyl is substituted by one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 selected from List A.

[0139] The term "alkylaryl" means an aryl group as defined above, which is substituted with an alkyl group as defined above, the aryl moiety being attached to the remainder of the molecule. Each of the aryl and alkyl moieties of the group may take any of the broadest or preferred meanings described above. "Substituted alkylaryl" means that one or more hydrogen atoms of the alkyl or aryl moiety of the group (such as one to a maximum number of hydrogen atoms bonded to the alkylaryl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the alkylaryl group is substituted by one or more, such as 1, 2, or 3, such as 1 or 2, such as 1 selected from List A.

[0140] The term "arylalkyl" means an alkyl group as defined above, which is substituted with an aryl group as defined above, the alkyl portion being attached to the remainder of the molecule. Each of the aryl and alkyl portions of the group may take any of the broadest or preferred meanings described above. "Substituted arylalkyl" means that one or more hydrogen atoms of the alkyl or aryl portion of the group (such as one to a maximum number of hydrogen atoms bonded to the arylalkyl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the arylalkyl group is substituted by one or more, such as 1, 2, or 3, such as 1 or 2, such as 1 selected from List A.

[0141] The term "alkylheteroaryl" means a heteroaryl group as defined above, which is substituted with an alkyl group as defined above, the heteroaryl moiety being attached to the remainder of the molecule. Each of the heteroaryl and alkyl moieties of the group may take any of the broadest or preferred meanings described above. "Substituted alkylheteroaryl" means that one or more hydrogen atoms of the alkyl or heteroaryl moiety of the group (such as one to a maximum number of hydrogen atoms bonded to the alkylheteroaryl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the alkylheteroaryl group is substituted by one or more, such as 1, 2, or 3, such as 1 or 2, such as 1 selected from List A.

[0142] The term "heteroarylalkyl" means an alkyl group as defined above, which is substituted with a heteroaryl group as defined above, the alkyl portion being attached to the remainder of the molecule. Each of the aryl and alkyl portions of the group may take any of the broadest or preferred meanings described above. "Substituted heteroarylalkyl" means that one or more hydrogen atoms of the alkyl or heteroaryl portion of the group (such as one to a maximum number of hydrogen atoms bonded to the heteroarylalkyl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the heteroarylalkyl group is substituted by one or more, such as 1, 2, or 3, such as 1 or 2, such as 1 selected from List A.

[0143] The term "alkyl heterocyclic group" means a heterocyclic group as defined above, which is substituted with an alkyl group as defined above, and the heteroaryl moiety is attached to the remainder of the molecule. Each of the heterocyclic and alkyl moieties of the group may take any of the broadest or preferred meanings described above. "Substituted alkyl heterocyclic group" means that one or more hydrogen atoms of the alkyl or heteroaryl moiety of the group (such as one to a maximum number of hydrogen atoms bonded to the alkyl heterocyclic group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the alkyl heterocyclic group is substituted by one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 selected from List A.

[0144] The term "heterocyclic alkyl" means an alkyl group as defined above, which is substituted with a heterocyclic group as defined above, the alkyl portion being attached to the remainder of the molecule. Each of the heterocyclic and alkyl portions of the group may take any of the broadest or preferred meanings described above. "Substituted heterocyclic alkyl" means that one or more hydrogen atoms of the alkyl or heterocyclic portion of the group (such as one to a maximum number of hydrogen atoms bonded to the heterocyclic alkyl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the heterocyclic alkyl is substituted by one or more, such as 1, 2, or 3, such as 1 or 2, such as 1 selected from List A.

[0145] The terms "organic sulfuric acid" or "sulfate ester" refer to compounds of the formula R-OSO2-OH, where R is a hydrocarbon or heteroalkyl group, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclic, or heterocyclic alkyl groups (all as defined above, whether in the broadest or preferred respect). The term "sulfate" is used when the group is deprotonated. The sulfate ester group can be protonated or deprotonated depending on the pH (in anionic amphiphiles as defined below, the sulfonic acid group is typically deprotonated at physiological pH).

[0146] The term "sulfonic acid" or "sulfonate ester" refers to a compound of the formula R-SO2-OH, where R is a hydrocarbon or heteroalkyl group, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclic, or heterocyclic alkyl groups (all as defined above, whether in the broadest or preferred respect). The term "sulfonate group" is used when the group is deprotonated. The sulfonate ester group can be protonated or deprotonated depending on the pH (in anionic amphiphiles as defined below, the sulfonate ester group is typically deprotonated at physiological pH).

[0147] The term "carboxylic acid" or "carboxylic acid ester" refers to a compound of the formula R-CO2H, where R is a hydrocarbon or heteroalkyl group, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclic, or heterocyclic alkyl groups (all as defined above, either in the broadest or preferred respect). The term "carboxylic acid group" is used when the group is deprotonated. Depending on pH, the carboxylic acid can be protonated or deprotonated (in anionic amphiphiles as defined below, the carboxylic acid group is typically protonated at acidic pH and deprotonated at neutral or basic pH).

[0148] The term "dicarboxylic acid" or "dicarboxylic acid ester" refers to a compound of the formula HO₂C-R'-CO₂H, where R' is an alkylene or alkenylene group (all as defined above, either in the broadest or preferred sense). When this group is deprotonated, the term "dicarboxylic acid ion" is used. Depending on pH, dicarboxylic acids can be protonated or deprotonated (in anionic amphiphiles as defined below, the dicarboxylic acid group is typically protonated at acidic or neutral pH and deprotonated at alkaline pH).

[0149] The term "hydroxycarboxylic acid" or "hydroxycarboxylic acid ester" refers to a compound of the formula R-CO2H, where R is a hydrocarbon or heteroalkyl group, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclic, or heterocyclic alkyl groups (all as defined above, either in the broadest or preferred aspects), which are substituted by one or more (preferably 1 to 5, such as 1, 2, or 3) hydroxyl groups. When this group is deprotonated, the term "hydroxycarboxylic acid ion" is used. Hydroxycarboxylic acids can be protonated or deprotonated depending on pH (in anionic amphiphiles as defined below, the carboxylic acid group is typically protonated at acidic pH and deprotonated at neutral or basic pH).

[0150] Depending on the context, as used herein, the term "ester" means a divalent bond of the structure -C(=O)O- or -OC(=O)- (where each end is attached to the remainder of the molecule), where both ends are attached to the remainder of the molecule, or a compound having the structure RC(O)O-R' (including its isomerically arranged structure R-OC(O)-R', unless otherwise specified), where R and R' are each independently a hydrocarbon or heteroalkyl group, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclic, or heterocyclic alkyl groups (all as defined above, whether in the broadest or preferred aspect). When the term refers to a substituent attached to the remainder of the molecule, the ester moiety may have the structure RC(O)O- or R-OC(O)-, where R is as defined above. In one embodiment, each of the two ends of the ester structure is covalently linked to the C atom of the same organic group or two separate organic groups (e.g., alkylene groups as additional components of the linking group).

[0151] As used herein, the term "thioester" refers to a divalent bond in the structure -C(=S)O-, -C(=O)S-, -SC(=O)-, or -OC(=S)-, with one end attached to a carbon atom and the other end attached to an oxygen or sulfur atom.

[0152] The term "phosphate ester" refers to a compound of the formula RO-P(=O)(OH)2, where R is a hydrocarbon or heteroalkyl group, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclic, or heterocyclic alkyl groups (all as defined above, whether in the broadest or preferred respect). Depending on pH, the phosphate ester group may be protonated or deprotonated (in anionic amphiphiles as defined below, the phosphate ester group is typically deprotonated at physiological pH).

[0153] The term "phosphonate" refers to a compound of the formula RP(=O)(OH)2, where R is a hydrocarbon or heteroalkyl group, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclic, or heterocyclic alkyl groups (all as defined above, whether in the broadest or preferred respect). Depending on pH, the phosphonate group may be protonated or deprotonated (in anionic amphiphiles as defined below, the phosphonate group is typically deprotonated at physiological pH).

[0154] "Halogen" refers to fluorine (-F), chlorine (-Cl), bromine (-Br), or iodine (-I).

[0155] "Amine" refers to the group –NR2, where each R is a hydrocarbon or heteroalkyl group, such as alkyl, alkenyl, ynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclic, or heterocyclic alkyl groups (all as defined above, whether in the broadest or preferred aspect), and preferably an alkyl group, such as C 1-6 Alkyl groups. When both groups R are hydrogen, the amine group is a primary amine group. When one R is hydrogen and the other R is different from hydrogen, the amine group is a secondary amine group. When neither group R is different from hydrogen, the amine group is a tertiary amine group.

[0156] Quaternary ammonium salts contain the –N group. + The compound R3, wherein each R is a hydrocarbon or heteroalkyl group, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclic, or heterocyclic alkyl (all as defined above, whether in the broadest or preferred aspect), and preferably an alkyl group, such as C 1-6 Alkyl groups. Unlike some amines, as defined above, which protonate only at specific pH values, quaternary ammonium salts carry a constitutive positive charge at all pH values ​​(as defined herein).

[0157] "Hydroxy" refers to the group – OH. "Mercapto" refers to the group – SH. "Nitro" refers to the group – NO2.

[0158] "Ether" means an oxygen atom attached to two hydrocarbon or heteroalkyl groups (such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclic, or heterocyclic alkyl groups (all as defined above, whether in the broadest or preferred aspect)). An ether can be a cyclic ether, wherein the two hydrocarbon groups together form a ring, and may include a dioxacyclopentyl group.

[0159] "Thioether" refers to a divalent bond of the formula –S–, where both connecting parts are via sulfur atoms, or a group of the formula –SR, where R is C. 1-10 Alkyl groups.

[0160] "Disulfide" refers to a divalent bond of the formula –S–S–, where one part is attached to the first sulfur atom and the other is attached to the second sulfur atom.

[0161] "Amide" refers to the group –C(=O)NR(R'), where R and R' are each independently a hydrogen or hydrocarbon or heteroalkyl group, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclic, or heterocyclic alkyl groups (all as defined above, either in the broadest or preferred respect), and preferably an alkyl group, such as C 1-6 Alkyl group; or a divalent bond of the formula –C(=O)NR–, wherein R is hydrogen or hydrogen or a hydrocarbon or heteroalkyl group, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclic, or heterocyclic alkyl group (all as defined above, whether in the broadest or preferred aspect), one portion of which is attached to the remainder of the molecule via a carbon atom and the other via a nitrogen atom.

[0162] "Thioamide" refers to a divalent bond of the formula –C(=S)NR–, where R is hydrogen or C. 1-6 An alkyl group, one part of which is attached to the rest of the molecule via a carbon atom and the other part of which is attached to the rest of the molecule via a nitrogen atom.

[0163] "Hydroxyamide" means the group –C(=O)O-NR(R'), where R and R' are each independently a hydrogen or hydrocarbon or heteroalkyl group, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclic, or heterocyclic alkyl groups (all as defined above, whether in the broadest or preferred respect).

[0164] "Sulfonyl" refers to the group –S(=O)2R, where R is C 1-13 Alkyl or C 2-3 Alkenyl groups (all as defined above, whether in the broadest or preferred sense), or divalent bonds of the formula –S(=O)2–, wherein each part is connected to the rest of the molecule via a sulfur atom.

[0165] "Sulfanamide" refers to the group –S(=O)2NRR', where R and R' are each independently a hydrogen or hydrocarbon or heteroalkyl group, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclic, or heterocyclic alkyl groups (all as defined above, whether in the broadest or preferred aspect), and preferably an alkyl group, such as C 1-30 Alkyl groups.

[0166] "Carbamate" refers to the group –OC(=O)NRR', where R and R' are each independently a hydrogen or hydrocarbon or heteroalkyl group, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclic, or heterocyclic alkyl groups (all as defined above, whether in the broadest or preferred aspect), and preferably an alkyl group, such as C 1-6 Alkyl groups.

[0167] "Amidine" signifies the group –C(=NR)NR'R", where R, R', and R" are each independently a hydrogen or hydrocarbon or heteroalkyl group, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclic, or heterocyclic alkyl groups (all as defined above, either in the broadest or preferred respect), and preferably an alkyl group, such as C 1-6 Alkyl groups.

[0168] “Guidin” means the group –NR-C(=NR')NR”R”' or =NC(NRR')(NR”R”'), wherein R, R', R”, and R”' are each independently a hydrogen or hydrocarbon or heteroalkyl group, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclic, or heterocyclic alkyl groups (all as defined above, whether in the broadest or preferred aspect), and preferably an alkyl group, such as C 1-6 Alkyl groups.

[0169] When protonated basic nitrogen atoms are involved, the above definitions can be modified according to normal chemical nomenclature by replacing the suffix "-ium". For example, guanidinium is a protonated guanidine, ammonium is a protonated ammonia or a protonated primary, secondary, or tertiary amine, imidazoleium is a protonated imidazole, pyridinium is a protonated pyridine, amidineium is a protonated amidine, and piperazineium is a protonated piperazine.

[0170] "Carbohydrates" refers to carbohydrates with an empirical formula C m (H2O) n The compound, wherein m may be different from or the same as n. The term "carbohydrate residue" or "carbohydrate moiety" defines a residue attached to another atom, wherein a 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 a D-configuration or an L-configuration. Furthermore, the monosaccharide moiety may be an aldose or a ketose moiety. Suitably, the monosaccharide moiety may have 3 to 8, preferably 4 to 6, more preferably 5 or 6 carbon atoms. In one embodiment, the monosaccharide moiety is a hexose moiety (i.e., it has 6 carbon atoms), examples of which include hexaloses such as glucose, galactose, allose, adroose, mannose, gulose, idose, and tarose, as well as hexketoses such as fructose and sorbose. Preferably, the hexose moiety is a glucose moiety.

[0171] In another embodiment, the monosaccharide moiety is a pentose moiety (i.e., it has 5 carbon atoms), such as ribose, arabinose, xylose, or lysolose. Preferably, the pentose moiety is an arabinose or xylose moiety.

[0172] In another embodiment, the carbohydrate can be a higher sugar (i.e., a disaccharide or oligosaccharide) comprising more than one monosaccharide moiety linked together by glycosidic bonds. When the monosaccharide moiety is a hexose moiety, the glycosidic bond can be a 1-α,1'-α glycosidic bond, a 1,2'-glycosidic bond (which can be a 1-α2' or 1'-β-2' glycosidic bond), a 1,3'-glycosidic bond (which can be a 1-α-3' or 1-β-3' glycosidic bond), a 1,4'-glycosidic bond (which can be a 1-α-4' or 1-β-4' glycosidic bond), a 1,6'-glycosidic bond (which can be a 1-α-6' or 1-β-6' glycosidic bond), or any combination thereof. In one embodiment, the higher sugar comprises two monosaccharide units (i.e., a disaccharide). Examples of suitable disaccharides include maltose, isomaltose, isomaltulose, lactose, sucrose, cellobiose, aspergillus niger, kosperidose, trehalose, and trehalulose. In another embodiment, the higher sugar comprises 3 to 10 monosaccharide units (i.e., oligosaccharides) in the chain, which may be branched or unbranched. Preferably, the oligosaccharide comprises 3 to 8, more preferably 3 to 6 monosaccharide units. Examples of suitable oligosaccharides include maltodextrin, maltotriose, maltotetraose, maltopentose, maltohexaose, maltoheptaose, melitriose, cellotriose, cellotetraose, cellopentose, cellohexaose, and celloheptaose.

[0173] "List A" replaces the basic choice C. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 Alkyne, 6- to 14-membered (e.g., 6- to 10-membered) aryl, 3- to 14-membered (e.g., 5- or 6-membered) heteroaryl, 3- to 14-membered (e.g., 3- to 7-membered) cycloalkyl, 3- to 14-membered (e.g., 3- to 7-membered) heterocyclic, halogen, -CN, azide, -NO2, -OR', -N(R')2, -S(O) 0-2 R'、-S(O) 1-2 OR'、-OS(O) 1-2 R'、-OS(O) 1-2 OR'、-S(O) 1-2 N(R')2、-OS(O) 1-2 N(R')2、-N(R')S(O) 1-2 R'、-N(R')S(O) 1- 2OR'、-C(=X 1 )R'、-C(=X 1 )X 1 R'、-X 1 C(=X 1 )R' and -X 1 C(=X 1 )X 1 The group consisting of R', where X 1Each R' is independently selected from O, S, NH and N(CH3); and each R' is independently selected from H and C. 1-4 Alkyl, C 2-4 alkenyl, C 2-4 The group consisting of alkynyl, 5- or 6-membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl, and 5- or 6-membered heterocyclic groups, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclic groups is optionally substituted by one, two, or three substituents, said substituents being independently selected from C10. 1-3 Alkyl, halogen, -CF3, -CN, azide, -NO2, -OH, -O(C) 1-3 alkyl), -S(C 1-3 Alkyl groups, -NH2, -NH(C) 1-3 alkyl), -N(C) 1-3 Alkyl)2、-NHS(O)2(C 1-3 Alkyl group), -S(O)2NH 2-z (C 1-3 alkyl) z -C(=O)OH, -C(=O)O(Cl) -3 Alkyl group), -C(=O)NH 2-z (C 1-3 alkyl) z -NHC(=O)(C1-3 alkyl), -NHC(=NH)NH z-2 (C1-3 alkyl) z and -N(C 1-3 alkyl)C(=NH)NH 2-z (C 1-3 alkyl) z A group consisting of each z independently being 0, 1, or 2 and each C 1-3 Alkyl groups are independently methyl or ethyl; A1, composed of C 1-3 Alkyl, phenyl, halogen, -CF3, -OH, -OCH3, -SCH3, -NH2- z (CH3) z It is composed of -C(=O)OH and -C(=O)OCH3, where z is 0, 1, or 2 and C 1-3 The alkyl group is methyl, ethyl, propyl, or isopropyl. In some embodiments, the substituents in list A are selected from list A2, which consists of methyl, ethyl, propyl, isopropyl, halogens (such as F, Cl, or Br), and -CF3.

[0174] Particles

[0175] In one aspect, the present invention provides functionalized particles as described herein. Functionalized particles are capable of delivering payloads (such as nucleic acid payloads) to a target. In use, the particles act to stabilize and encapsulate the payload (such as nucleic acid) so that it can be delivered into cells, while simultaneously promoting its uptake into cells and release into the cytosol. In the context of this disclosure, the term "particle" refers to a structured entity formed of molecules or molecular complexes, particularly particle-forming compounds. In some embodiments, the particle is a nucleic acid-containing particle, such as a particle containing DNA, RNA, or a mixture thereof.

[0176] In some embodiments, the particles contain a coating (e.g., one or more layers or thin layers) made of one or more types of amphiphilic substances (e.g., amphiphilic lipids). In this context, the term "amphiphilic substance" means a substance that has both hydrophilic and lipophilic properties. The coating may also contain other substances that are not necessarily amphiphilic (e.g., additional lipids). Thus, the particles can be monolayer or multilayer structures, wherein the substances constituting the one or more layers or thin layers comprise one or more types of amphiphilic substances (particularly selected from the group consisting of amphiphilic lipids) optionally in combination with other substances that are not necessarily amphiphilic (e.g., additional lipids). In some embodiments, the term "particle" refers to a micrometer or nanometer-scale structure, such as a dense structure at the micrometer or nanometer scale.

[0177] According to this disclosure, the term "particle" includes nanoparticles. The term "nanoparticle" refers to a nanoscale particle containing at least one particle-forming agent (e.g., at least one cationic lipid or cationic ionizable lipid or cationic polymer), wherein all three external dimensions of the particle are at the nanoscale, i.e., at least about 1 nm and less than about 1000 nm. Preferably, the size of the particle is its diameter.

[0178] In some embodiments, the particles described herein have a size (such as 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 1200 nm, at most about 1100 nm, at most about 1000 nm, at most about 950 nm). The range is approximately 20 to 1500 nm, such as approximately 30 to 1200 nm, approximately 40 to 1100 nm, approximately 50 to 1000 nm, or approximately 100 to 500 nm, such as approximately 10 to 1000 nm, 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, 50 to 250 nm, or 60 to 200 nm. The particle is in the range of nm, 70 to 150 nm, or 80 to 150 nm. In some embodiments, the particles described herein have a size (such as diameter) in the range of about 40 nm to about 200 nm, such as about 50 nm to about 180 nm, about 60 nm to about 160 nm, about 80 nm to about 150 nm, or about 80 nm to about 120 nm.

[0179] In some embodiments, the particles described herein have an average diameter that, in some embodiments, ranges from about 50 nm to about 1000 nm, about 50 nm to about 800 nm, about 50 nm to about 700 nm, about 50 nm to about 600 nm, about 50 nm to about 500 nm, about 50 nm to about 450 nm, about 50 nm to about 400 nm, about 50 nm to about 350 nm, about 50 nm to about 300 nm, about 50 nm to about 250 nm, about 50 nm to about 200 nm, about 100 nm to about 1000 nm, about 100 nm to about 800 nm, about 100 nm to about 700 nm, about 100 nm to about 600 nm, about 100 nm to about 500 nm, about 100 nm to about 450 nm, about 100 nm to about 400 nm, about 100 nm to about 350 nm, about 100 nm to about 300 nm, about 100 nm to about 250 nm, about 100 nm to about 100 nm to 250 nm, about 100 nm to about 100 nm to 10 ... nm to about 200 nm, about 150 nm to about 1000 nm, about 150 nm to about 800 nm, about 150 nm to about 700 nm, about 150 nm to about 600 nm, about 150 nm to about 500 nm, about 150 nm to about 450 nm, about 150 nm to about 400 nm, about 150 nm to about 350 nm, about 150 nm to about 300 nm, about 150 nm to about 250 nm, about 150 nm to about 200 nm, about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 200 nm to about 700 nm, about 200 nm to about 600 nm, about 200 nm to about 500 nm, about 200 nm to about 450 nm, about 200 nm to about 400 nm, about 200 nm to about 350 nm, about 200 nm to about 300 nm, about 200 nm to about 250 nm nm, or about 80 to about 150 nm. In some embodiments, the particles described herein have an average diameter ranging from about 40 nm to about 200 nm, such as about 50 nm to about 180 nm, about 60 nm to about 160 nm, about 80 nm to about 150 nm, or about 80 nm to about 120 nm.

[0180] In some implementations, nucleic acids can be non-covalently associated with particles. In some implementations, nucleic acids can adhere to the outer surface of the particles (surface nucleic acids) and / or can be contained within the particles (encapsulated nucleic acids).

[0181] The N / P ratio gives the ratio of the number of nitrogen groups in the lipid to the number of phosphate groups in the nucleic acid. It is related to the charge ratio because nitrogen atoms (depending on pH) are generally positively charged and phosphate groups are negatively charged. In the presence of charge balance, the N / P ratio depends on pH. Lipid formulations are typically formed with N / P ratios greater than four up to twelve because positively charged nanoparticles are considered favorable for transfection. In this case, the nucleic acid is considered to bind completely to the nanoparticles.

[0182] lipid nanoparticles

[0183] In one embodiment of this disclosure, the functionalized particles are lipid nanoparticles (LNPs). The LNPs and / or their lipid components may have adjuvant activity. Typically, the one or more particle-forming components in the LNPs comprise cationic lipids or cationic ionizable lipids, as defined and illustrated below.

[0184] In this disclosure, LNP can be understood as an oil-in-water emulsion, wherein the core material of the LNP is preferably in a liquid state and therefore has a melting point below body temperature. Thus, LNPs typically comprise a central lipid complex and optional loading, such as nucleic acids (e.g., RNA (such as mRNA), DNA, or mixtures thereof) embedded in a disordered, non-layered phase made of lipids. This contrasts with the structure of liposomes, which comprise monolayers or multilayers of vesicle particles, wherein the thin layer comprises a lipid bilayer surrounding an encapsulated aqueous cavity. In some cases, the pre-formed lipid particles and / or nucleic acid-lipid particles described herein are not liposomes.

[0185] Lipid nanoparticles (LNPs) can be obtained by combining a load (such as nucleic acid) with lipids. The lipids used for LNP formation typically do not form a layered (bilayer) phase in water under physiological conditions. LNPs typically do not contain or encapsulate an aqueous core. LNPs typically contain a lipid (or oily) core.

[0186] In some embodiments, the lipid nanoparticles described herein have an average size (such as diameter) ranging 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 1300 nm, at most about 1200 nm, at most about 1100 nm, at most about 1000 nm). (nm, up to about 950 nm, up to about 900 nm, up to about 850 nm, up to about 800 nm, up to about 750 nm, up to about 700 nm, up to about 650 nm, up to about 600 nm, up to about 550 nm, or up to about 500 nm)Such as about 50 nm to about 1000 nm, about 50 nm to about 800 nm, about 50 nm to about 700 nm, about 50 nm to about 600 nm, about 50 nm to about 500 nm, about 50 nm to about 450 nm, about 50 nm to about 400 nm, about 50 nm to about 350 nm, about 50 nm to about 300 nm, about 50 nm to about 250 nm, about 50 nm to about 200 nm, about 100 nm to about 1000 nm, about 100 nm to about 800 nm, about 100 nm to about 700 nm, about 100 nm to about 600 nm, about 100 nm to about 500 nm, about 100 nm to about 450 nm, about 100 nm to about 400 nm, about 100 nm to about 350 nm, about 100 nm to about 300 nm, about 100 nm to about 250 nm, about 100 nm to about 200 nm, about 150 nm to about 1000 nm nm, about 150 nm to about 800 nm, about 150 nm to about 700 nm, about 150 nm to about 600 nm, about 150 nm to about 500 nm, about 150 nm to about 450 nm, about 150 nm to about 400 nm, about 150 nm to about 350 nm, about 150 nm to about 300 nm, about 150 nm to about 250 nm, about 150 nm to about 200 nm, about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 200 nm to about 700 nm, about 200 nm to about 600 nm, about 200 nm to about 500 nm, about 200 nm to about 450 nm, about 200 nm to about 400 nm, about 200 nm to about 350 nm, about 200 nm to about 300 nm, or about 200 nm to about 250 nm.

[0187] Lipoplex

[0188] In one embodiment, the functionalized particles of this disclosure are lipid complexes (LPX). In such particles, the one or more particle-forming components comprise cationic lipids or cationic ionizable lipids. Lipid complexes (LPX) are electrostatic complexes that are typically formed by mixing pre-formed particles containing cationic lipids or cationic ionizable lipids with an anionic load (such as nucleic acids). The resulting lipid complexes have a different internal molecular arrangement resulting from the transformation from a liposome structure to a dense nucleic acid-lipid complex.

[0189] Polymer complex

[0190] In one embodiment, the functionalized particles of this disclosure are polymer complexes (PLX). In such particles, the one or more particle-forming components comprise cationic polymers, as defined and exemplified below. Typically, such cationic polymers are capable of electrostatically agglomerating negatively charged loads (such as nucleic acids) into the particles. As described below, polymer complexes may also contain anionic polymers. Polymer complexes may also contain neutral (e.g., hydrophilic) polymers, such as stealth polymers (e.g., PEG, pSar, pAEEA, etc.) as described below.

[0191] Esterified polymer complex

[0192] In one embodiment, the functionalized particles of this disclosure are lipopolymer complexes (LPLX). In such particles, the one or more particle-forming components comprise cationic polymers, as defined and exemplified below. Typically, the cationic polymers used for LPLX formation comprise hydrophobic portions to facilitate interaction with the lipid components of the LPLX (e.g., Viromers), as further described below. The LPLX may also contain neutral (e.g., hydrophilic) polymers, such as stealth polymers (e.g., PEG, pSar, pAEEA, etc.) as described below. Lipidopolymer complexes also contain lipids, as defined and exemplified below.

[0193] Functionalized particles

[0194] This invention relates to functionalized particles. As described herein, “functionalized” particles include, bind to, or interact with compounds comprising, binding to, or interacting with an anti-IgD portion that binds to IgD on target cells. Thus, “functionalized particles” can be understood as particles exhibiting preferential interaction with target cells that express or exhibit IgD (which are preferentially recognized by the major targeting portion of the particle (i.e., the portion capable of binding to IgD on the target cell)). Therefore, functionalized particles provide targeted delivery of a payload / active ingredient (such as a nucleic acid) to specific target cells.

[0195] Functionalized particles enable the application of payloads (such as nucleic acid payloads) together with one or more delivery media that protect the payload from degradation, maximize delivery to target cells, and minimize exposure to non-target cells. Such delivery media can composite or encapsulate the payload and comprise a range of materials, including polymers, lipids, and mixtures thereof. In some embodiments, such delivery media can form particles with the payload. Functionalized particles can be functionalized lipid particles, such as functionalized LNPs or functionalized LPXs. Functionalized particles can be functionalized polymer complexes (PLXs). Functionalized particles can be functionalized lipopolymer complexes (LPLXs).

[0196] The functionalized particles of this invention typically comprise:

[0197] (a) One or more particulate-forming components, as defined herein;

[0198] (b) payloads, such as nucleic acid payloads; and

[0199] (c) The portion that can bind to immunoglobulin D (IgD).

[0200] In one implementation, the functionalized particles comprise:

[0201] (a) One or more particulate-forming components, as defined herein;

[0202] (b) Loading, such as nucleic acid loads;

[0203] (c) A linker compound comprising:

[0204] (i) The portion capable of incorporating linker compounds into the particles, and

[0205] (ii) the first interaction part; and

[0206] (d) Docking compounds, which include:

[0207] (i) the second interaction component, and

[0208] (ii) The portion that can bind to IgD;

[0209] The first and second interacting parts can combine with each other.

[0210] In one implementation, the functionalized particles comprise:

[0211] (a) One or more particulate-forming components, as defined herein;

[0212] (b) Loading, such as nucleic acid loads;

[0213] (c) Linker compounds of formula (A):

[0214] L‐X1‐P‐X2‐B1 (A); and

[0215] (d) Docking compounds of formula (I):

[0216] B2-X3-B3 (I)

[0217] in:

[0218] P either does not exist or contains a polymer;

[0219] L contains a portion capable of incorporating the compound into the particles, which is attached to B1 when P is absent, or to the first end of polymer P when P is present.

[0220] B1 contains a portion that can bind to B2. When P is absent, a portion of B1 is attached to L, or when P is present, it is attached to the second end of polymer P.

[0221] X1 and X2 are either independent or connected parts;

[0222] B2 contains a portion that can combine with B1;

[0223] X3 is missing or is a connection part; and

[0224] B3 contains a portion that can bind to IgD.

[0225] In some exemplary functionalized particles, as described herein, the particles comprise a compound of formula (A'), wherein portion B' of the compound of formula (A') is a portion that binds to IgD on target cells. In such embodiments, the compound of formula (A') functionalizes the particles such that, in use, a link can be established between portion B' of the compound of formula (A') and IgD, enabling the payload contained in the loaded lipid particles to be delivered to target cells expressing IgD or presenting IgD on their surface.

[0226] In some exemplary functionalized particles, a particle comprising a linker compound as defined herein is brought into contact with a docking compound as defined herein, such that the second interacting portion of the docking compound of formula (I) interacts with the first interacting portion of the linker compound.

[0227] In some exemplary functionalized particles, a particle comprising a compound of formula (A) as described herein is contacted with a compound of formula (I) as defined herein, such that portion B2 of the compound of formula (I) interacts with portion B1 of the compound of formula (A). Thus, in some embodiments, the compound of formula (I) interacts with or binds to a loaded lipid particle as described herein.

[0228] In this context, the terms "interact with" and "bind with" are used interchangeably. A docking compound (e.g., a compound of formula (I)) may interact with or bind to a linker compound (such as a compound of formula (A)) covalently or non-covalently (preferably non-covalently). In such embodiments, the interaction between the linker compound and the docking compound functionalizes the particle such that, in use, a link can be established between the docking compound and IgD (e.g., IgD on a target cell) to enable the delivery of a payload (such as nucleic acid) contained in the functionalized particle to target cells expressing IgD or presenting IgD on their surface.

[0229] In one embodiment, portion B1 comprises an antibody, an antibody-like molecule, or a VHH, and portion B2 is a peptide.

[0230] In one embodiment, portion B2 comprises an antibody, an antibody-like molecule, or a VHH, and portion B1 is a peptide.

[0231] In one embodiment, portion B3 is a single-domain antibody (sdAb) capable of binding IgD. In one embodiment, the sdAb is a variable heavy chain domain antibody (VHH), a variable heavy chain (VH) domain antibody, or a variable neoantigen receptor antibody (VNAR). In one embodiment, the sdAb is a VHH.

[0232] In one embodiment, the compound of formula B2-X3-B3 comprises a peptide or polypeptide.

[0233] In one implementation, the portion capable of binding to IgD comprises an antibody or antibody-like molecule.

[0234] In one embodiment, portion B1 contains a peptide tag and portion B2 contains a portion capable of binding to the peptide tag.

[0235] In one embodiment, portion B2 contains a peptide tag and portion B1 contains a portion capable of binding to the peptide tag.

[0236] In one implementation, the portion capable of binding to the peptide tag comprises an antibody or antibody-like molecule.

[0237] In one implementation, the peptide tag includes an ALFA-tag, as defined below.

[0238] In one embodiment, portion B1 of the compound of formula (A) comprises a peptide tag, and portion B2 of the compound of formula (I) binds to the compound of formula (A) via the peptide tag. In this embodiment, the peptide tag preferably comprises an ALFA-tag. In this embodiment, portion B2 of the compound of formula (I) is preferably a VHH capable of binding to an ALFA-tag.

[0239] In one embodiment, portion B2 of the compound of formula (I) comprises a peptide tag, and portion B1 of the compound of formula (A) binds to the compound of formula (I) via the peptide tag. In this embodiment, the peptide tag preferably comprises an ALFA-tag. In this embodiment, portion B1 of the compound of formula (A) is a VHH capable of binding to the ALFA-tag.

[0240] load

[0241] Functionalized lipid particles, as defined herein, contain a payload. In some embodiments, the payload contains a therapeutic or diagnostic component. 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.

[0242] The payload may be a therapeutic agent, such as a pharmaceutically active agent. Examples of pharmaceutically active agents are known to those skilled in the art and are provided herein. The therapeutic agent may also optionally contain a detectable marker, as defined below.

[0243] Therefore, according to some implementation schemes, the agents and methods described herein are used for targeted therapy. This is achieved by using a payload containing one or more pharmaceutically active agents (e.g., drugs for radiotherapy or radioisotopes).

[0244] The term "pharmaceutical active agent" refers to any agent, such as a compound or cell, that is therapeutically effective when administered to an individual. The term "pharmaceutical active agent" also includes any agent that alters, preferably cures, alleviates, or partially blocks the clinical presentation of a given disease and its complications in a therapeutic intervention involving the administration of said agent.

[0245] In some embodiments, the payload is a nucleic acid, such as pharmaceutically active RNA (e.g., mRNA) or DNA, as defined in more detail below. In some embodiments, the pharmaceutically active agent comprises a pharmaceutically active peptide or protein, as defined in more detail below.

[0246] In some embodiments, the payload comprises nucleic acids, as defined in more detail below. Typically, 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 biomolecules encoded by the nucleic acid, such as peptides or proteins. In some embodiments, the nucleic acid is a nucleic acid encoding an antigen receptor. In these embodiments, the target cells can be immune cells or immune effector cells.

[0247] In some implementations, the payload is a gene-editing reagent or tool (e.g., a transposon (such as Sleeping Beauty or Piggy Bac) or a CRISPR / Cas (or related)-based system). Such tools for genome integration / editing (e.g., transposases, gene-editing tools such as CRISPR / Cas9) can be delivered as proteins or encoding nucleic acids (DNA or RNA). For example, a payload may contain one or more nucleic acids (e.g., RNA or mRNA) encoding a transposon (e.g., Sleeping Beauty transposase, such as SB100X) and one or more nucleic acid transposons (e.g., DNA). In such cases, the transposon (e.g., DNA transposon) typically encodes a target gene for insertion into the genome. For example, a payload may contain one or more nucleic acids (e.g., RNA or mRNA) encoding a Cas endonuclease (e.g., Cas9) and gRNA. A payload may include a peptide or protein containing the function of a Cas endonuclease (e.g., Cas9) and one or more nucleic acids (e.g., RNA, mRNA, DNA, or mixtures thereof) containing gRNA. Cas is an enzyme that uses a CRISPR sequence as a guide to recognize and cut specific DNA strands complementary to the CRISPR sequence. Together with the CRISPR sequence, the Cas enzyme forms the basis of a technology called CRISPR-Cas, which can be used to edit genes in organisms. This editing process has a variety of applications, including basic biological research, the development of biotechnology products, and the treatment of diseases.

[0248] CRISPR / Cas is a target-specific technology that introduces gene knockout or knock-in based on the double-strand repair pathway. The targeting specificity of CRISPR-Cas is determined by a 20-nt sequence at the 5' end of the guide RNA (gRNA). The desired target sequence must be located as a short DNA sequence, typically 2-6 base pairs in length, preceding the prespacer adjacent motif (PAM) following the DNA region targeted for cleavage by CRISPR-Cas. The PAM is required for Cas nuclease cleavage and is typically found 3-4 nucleotides downstream of the cleavage site. After the gRNA pairs with the target base, Cas mediates a double-strand break approximately 3-nt upstream of the PAM.

[0249] In some embodiments, the payload contains compounds that can be used in radiotherapy and / or chemotherapy. In some embodiments, the payload contains chemotherapeutic compounds.

[0250] Chemotherapy is a type of cancer treatment that uses one or more anticancer drugs (chemotherapeutic agents), usually as part of a standardized chemotherapy regimen. The term chemotherapy has come to mean the non-specific use of endotoxins to inhibit mitosis. It does not include more selective agents that block extracellular signals (signal transduction). The development of therapies with specific molecular or gene targets that inhibit growth-promoting signals from classical endocrine hormones (primarily estrogen for breast cancer and androgens for prostate cancer) is now known as hormone therapy. In contrast, other growth signaling inhibitions, such as those associated with receptor tyrosine kinases, are called targeted therapies.

[0251] Traditional chemotherapy agents are cytotoxic by interfering with cell division (mitosis), but cancer cells vary greatly in their susceptibility to these agents. To a large extent, chemotherapy can be considered a way of damaging or stressing cells, and if it induces apoptosis, it can lead to cell death.

[0252] Chemotherapy agents include alkylating agents, antimetabolites, antimicrotubule agents, topoisomerase inhibitors, and cytotoxic antibiotics.

[0253] Alkylating agents have the ability to alkylate many molecules, including proteins, RNA, and DNA. Subtypes of alkylating agents include nitrogen mustards, nitrosoureas, tetrazides, aziridines, cisplatin and its derivatives, as well as non-classical alkylating agents. Nitrogen mustards include nitrogen mustard, cyclophosphamide, melphalan, chlorambucil, ifosfamide, and busulfan. Nitrosoureas include N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), formustine, and streptozotocin. Tetrazides include dacarbazine, mitoxazolamide, and temozolomide. Aziridines include thiotepa, mytomycin, and diaziquone (AZQ). Cisplatin and its derivatives include cisplatin, carboplatin, and oxaliplatin. They impair cellular function by forming covalent bonds with amino, carboxyl, thiol, and phosphate groups in biologically important molecules. Non-classical alkylating agents include procarbazine and hexamethylmelamine. In a particularly preferred embodiment, the alkylating agent is cyclophosphamide.

[0254] Antimetabolites are a group of molecules that inhibit DNA and RNA synthesis. Many of them have structures similar to the building blocks of DNA and RNA. Antimetabolites resemble nucleosides or nucleosides but have altered chemical groups. These drugs exert their effects by blocking enzymes required for DNA synthesis or by being incorporated into DNA or RNA. Subtypes of antimetabolites include antifolates, fluoropyrimidines, deoxynucleoside analogs, and thiopurines. Antifolates include methotrexate and pemetrexed. Fluoropyrimidines include fluorouracil and capecitabine. Deoxynucleoside analogs include cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelabine, cladribine, clofarabine, and pentostatin. Thiopurines include thioguanine and mercaptopurine.

[0255] Anti-microtubule agents block cell division by inhibiting microtubule function. Vinca minor alkaloids inhibit microtubule formation, while taxanes inhibit microtubule depolymerization. Vinca minor alkaloids include vinorelbine, vindesine, and vinflunine. Taxanes include docetaxel (Taxotere) and paclitaxel (Taxotere).

[0256] 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, neomycin, merbarone, and azorubicin.

[0257] Cytotoxic antibiotics are a diverse group of drugs with different mechanisms of action. A common theme they share in their chemotherapeutic indications is that they disrupt cell division. The most important subgroups are anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, and arubicin) and bleomycin; other prominent examples include mitomycin C, mitoxantrone, and actinomycin.

[0258] In some embodiments, the payload is a detectable marker. As used herein, a “detectable marker” refers to a compound that allows for detection, for example, when present in cells, tissues, or organisms. One type of detectable marker contemplated within the context of this disclosure is a contrast provider. Different types of detectable markers are contemplated within the context of this disclosure and are described herein.

[0259] Therefore, according to some embodiments of this disclosure, the reagents and methods of this disclosure are used in imaging, particularly medical imaging. To identify the primary target, an imaging probe containing one or more detectable markers can be used as a payload. Specific examples of detectable markers for imaging probes are contrast-providing portions used in conventional imaging systems, such as MRI imaging constructs, spin labels, optical labels, ultrasound-responsive constructs, X-ray-responsive portions, radionuclides, (bio)luminescent materials, and FRET-type dyes. Exemplary detectable markers contemplated within the context of this disclosure include, but are not limited to, fluorescent molecules, such as autofluorescent molecules, molecules that fluoresce upon contact with a reagent, etc.; radiolabels; biotin, for example, detected by the binding of biotin to avidin; fluorescent tags; MRI imaging constructs containing paramagnetic metals; imaging reagents, etc. The radionuclide used for imaging can be, for example, selected from... 3 H, 11 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、 99 Tc, 110 In、 111 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, 201 Tland 203 Isotopes of the group consisting of Pb. Other elements and isotopes, such as those used in therapy, can also be used in imaging for certain applications.

[0260] The MRI-imaging component can be either paramagnetic ions or superparamagnetic particles. Paramagnetic ions can be elements 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, and Tl.

[0261] The X-ray response components include, but are not limited to, iodine, barium, and barium sulfate.

[0262] Furthermore, within the context of this disclosure, detectable markers also include peptides or polypeptides that can be detected by antibody binding (e.g., binding of an antibody to a detectable marker). In some embodiments, the detectable markers are small-sized organic PET and SPECT markers, such as... 18 F, 11 C or 123 I.

[0263] Nucleic acid

[0264] In some embodiments, the payload is nucleic acid. Preferably, the functionalized lipid particle compositions of this application contain RNA (such as mRNA) and / or DNA. Generally, the functionalized lipid particle compositions described herein comprise lipid particles encapsulating nucleic acids. The term "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term includes genomic DNA, cDNA, mRNA, recombinantly produced, and chemically synthesized molecules. In one embodiment, the nucleic acid is RNA. In one embodiment, the nucleic acid is mRNA. In one embodiment, the nucleic acid is DNA. In one embodiment, the active ingredient (e.g., which will be delivered to target cells to genetically modify the target cells and enable the target cells to express biomolecules (such as peptides or proteins encoded by nucleic acids)) comprises DNA, RNA, or mixtures thereof.

[0265] Nucleic acids can exist as single-stranded or double-stranded molecules and as linearly or covalently closed circular molecules. Nucleic acids can be isolated. According to this disclosure, the term "isolated nucleic acid" means that the nucleic acid is (i) amplified in vitro, for example, by polymerase chain reaction (PCR) for DNA or by in vitro transcription of RNA (using, for example, RNA polymerase), (ii) produced by clonal recombination, (iii) purified, for example by cutting and separation by gel electrophoresis, or (iv) synthesized, for example by chemical synthesis.

[0266] The term "nucleoside" refers to a compound that can be considered a nucleotide without a phosphate group. A nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), while a nucleotide consists of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine. Nucleic acids can include one or more modified nucleosides or nucleotides. Examples of modified nucleosides or nucleotides that can be incorporated into nucleic acids include N7-alkylguanine, N6-alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil, and N(1)-alkyl-uracil, such as N7-C1-4alkylguanine, N6-C1-4alkyl-adenine, 5-C1-4alkyl-cytosine, 5-C1-4alkyl-uracil, and N(1)-C1-4alkyl-uracil, preferably N7-methyl-guanine, N6-methyl-adenine, 5-methyl-cytosine, 5-methyl-uridine (m5U), pseudouridine (ψ), and N1-methyl-pseudouridine (m1Ψ).

[0267] RNA

[0268] In some embodiments of all aspects of this disclosure, the nucleic acid is RNA. According to this disclosure, the term "RNA" means a nucleic acid molecule comprising ribonucleotide residues. RNA typically contains naturally occurring nucleic acids such as adenosine (A), uridine (U), cytidine (C), and guanosine (G). In a preferred embodiment, the RNA contains all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of the β-D-furanose ribosyl group. RNA encompasses, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA (such as partially purified RNA), substantially pure RNA, synthetic RNA, recombinant RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alteration may refer to the addition of non-nucleotide material to the internal RNA nucleotides or to one or more ends of the RNA. It is also contemplated herein that the nucleotides in the RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the purposes of this disclosure, these altered / modified nucleotides (or modified nucleosides) may be referred to as analogs of naturally occurring nucleotides (nucleosides), and the corresponding RNA containing such altered / modified nucleotides or nucleosides (i.e., altered / modified RNA) may be referred to as analogs of naturally occurring RNA. If the content of ribonucleotide residues in a molecule is greater than 50% (e.g., 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, then the molecule contains “a majority of ribonucleotide residues”. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (regardless of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or their analogs). “RNA” includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), trans-amplifying RNA (taRNA), single-stranded RNA (ssRNA), dsRNA, repressive RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA), and immunostimulatory RNA (isRNA). In some embodiments, “RNA” refers to mRNA. The active ingredient can be mRNA, saRNA, taRNA, or a mixture thereof. The active ingredient is preferably mRNA. In some cases, the active ingredient is not siRNA.

[0269] In a preferred embodiment, the RNA comprises an open reading frame (ORF) encoding a peptide, polypeptide, or protein. The RNA may be capable of or configured to express the encoded peptide, polypeptide, or protein. For example, the RNA may be RNA that encodes and is capable of or configured to express a pharmaceutically active peptide or protein. In some embodiments, the RNA is capable of interacting with cellular translation mechanisms that allow the translation of the peptide or protein. The cell may produce the encoded peptide or protein intracellularly (e.g., in the cytoplasm), secrete the encoded peptide or protein, or produce it on its surface. Alternatively, the RNA may be non-coding RNA, such as antisense RNA, microRNA (miRNA), or siRNA.

[0270] mRNA

[0271] In preferred embodiments of all aspects of this disclosure, the nucleic acid is mRNA. According to this disclosure, the term "mRNA" means "messenger RNA" and includes "transcriptions" that can be generated using a DNA template. Typically, mRNA encodes peptides, polypeptides, or proteins. As established in the art, RNA (such as mRNA) typically contains a 5' untranslated region (5'-UTR), a peptide / polypeptide / protein coding region, and a 3' untranslated region (3'-UTR).

[0272] mRNA is single-stranded, but it can contain self-complementary sequences that allow the mRNA to partially fold and pair with itself to form a double helix.

[0273] According to this disclosure, "dsRNA" means double-stranded RNA and is RNA having two partially or completely complementary strands.

[0274] In a preferred embodiment of this disclosure, mRNA refers to an RNA transcript that encodes a peptide, polypeptide, or protein.

[0275] In some embodiments, the RNA encoding a peptide, polypeptide, or protein preferably 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, or at least 9,000 nucleotides), preferably up to 15,000, such as up to 14,000, up to 13,000, up to 12,000, up to 11,000, or up to 10,000 nucleotides.

[0276] In some implementations, RNA (such as mRNA) is produced by in vitro transcription or chemical synthesis. Preferably, RNA (such as mRNA) is produced by in vitro transcription using a DNA template. As used herein, the term “in vitro transcription” or “IVT” means that transcription (i.e., RNA production) is performed in a cell-free manner. That is, IVT does not use living / cultured cells, but uses transcription mechanisms extracted from cells (e.g., cell lysates or their isolated components, including RNA polymerases (preferably T7, T3, or SP6 polymerases)). In vitro transcription methods are known to those skilled in the art; see, for example, Molecular Cloning: A Laboratory Manual, 2nd ed., edited by J. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989. Furthermore, various in vitro transcription kits are available, for example, from Thermo Fisher Scientific (such as TranscriptAid). TM T7 kits, MEGAscript® T7 kits, MAXIscript®, New England BioLabs Inc. (such as HiScribe™ T7 kits, HiScribe™ T7 ARCA mRNA kits), Promega (such as RiboMAX™, HeLaScribe®, Riboprobe® systems), Jena Bioscience (such as SP6 or T7 transcription kits), and Epicentre (such as AmpliScribe™) are commercially available.

[0277] To provide modified RNA (such as mRNA), a correspondingly modified nucleotide, such as a modified naturally occurring nucleotide, a non-naturally occurring nucleotide, and / or a modified non-naturally occurring nucleotide, may be incorporated during synthesis (preferably in vitro transcription), or the modification may be performed in the mRNA after transcription and / or added to the mRNA. RNA (such as mRNA) may be modified. RNA (such as mRNA) may contain modified nucleotides or nucleosides, such as 5-methylcytosine, 5-methyluridine (m5U), pseudouridine (ψ), or N(1)-methyl-pseudouridine (m1ψ). One or more uridines in the RNA described herein may be replaced by a modified nucleoside. The modified nucleoside may be a modified uridine. RNA may contain a modified nucleoside replacing at least one uridine. Preferably, RNA may contain a modified nucleoside replacing each uridine (e.g., all uridines in the RNA are replaced by a modified nucleoside). The modified nucleoside may be independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyluridine (m5U). The modified nucleoside is preferably pseudouridine (ψ) or N1-methyl-pseudouridine (m1ψ).

[0278] In some embodiments, the RNA (such as mRNA) is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription from a suitable DNA template. The promoter used to control transcription can be any promoter of any RNA polymerase. Specific examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription is controlled by a T7 or SP6 promoter. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0279] In some embodiments of this disclosure, RNA (such as mRNA) is a "replicon RNA" (such as "replicon mRNA") or simply "replicon," particularly a "self-replicating RNA" (such as "self-replicating mRNA") or a "self-amplifying RNA" (or "self-amplifying mRNA"). Lipid particles containing RNA as described herein may contain mRNA, saRNA, taRNA, or mixtures thereof. Lipid particles containing RNA as described herein may contain mRNA encoding a replicase protein and one or more RNA molecules capable of being replicated or amplified by a replicase.

[0280] Repressive RNA

[0281] In some embodiments of all aspects of this disclosure, the nucleic acid is a repressive RNA.

[0282] As used herein, the term "repressive RNA" refers to RNA that selectively hybridizes to and / or is specific to a target mRNA, thereby inhibiting (e.g., reducing) its transcription and / or translation. Repressive RNAs include RNA molecules having a sequence having an antisense orientation relative to the target mRNA. Suitable repressive oligonucleotides typically vary in length from five to several hundred nucleotides, more typically being about 20 to 70 nucleotides or shorter, and even more typically about 10 to 30 nucleotides. Examples of repressive RNAs include antisense RNA, ribozymes, iRNA, siRNA, and miRNA. In some embodiments of all aspects of this disclosure, the repressive RNA is siRNA.

[0283] As used herein, the term "antisense RNA" refers to RNA that, under physiological conditions, hybridizes with DNA containing a specific gene or with the mRNA of said gene, thereby inhibiting the transcription of said gene and / or the translation of said mRNA.

[0284] The size of antisense RNA can range from 15 to 15,000 nucleotides, preferably 20 to 12,000, especially 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.

[0285] As used herein, “small interfering RNA” or “siRNA” refers to an RNA molecule preferably longer than 10 nucleotides, more preferably longer than 15 nucleotides, and most preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, capable of specifically binding to a portion of a target mRNA. This binding induces the cleavage or degradation of said portion of the target mRNA, thereby inhibiting gene expression of said target mRNA. The range of 19 to 25 nucleotides is the most preferred size for siRNA. Typically, siRNA comprises a single molecule in which two complementary portions are base-paired and covalently linked by a single-stranded “hairpin” region. Without being bound by any theory, it is assumed that the hairpin region of the siRNA molecule is cleaved within the cell by a “Dicer” protein (or its equivalent) to form two separate base-paired RNA molecules.

[0286] As used herein, “target mRNA” refers to an RNA molecule that is a target for downregulation. In some embodiments, the target mRNA comprises an ORF encoding a pharmacologically active peptide or polypeptide as specified herein. In some embodiments, the pharmacologically active peptide or polypeptide is a peptide or polypeptide whose expression (particularly increased expression, e.g., compared to expression in healthy subjects) is associated with a disease. In some embodiments, the target mRNA comprises an ORF encoding a pharmacologically active peptide or polypeptide whose expression (particularly increased expression, e.g., compared to expression in healthy subjects) is associated with a cancer.

[0287] According to this disclosure, siRNA can target any segment of approximately 19 to 25 consecutive nucleotides in any target mRNA sequence (“target sequence”). Techniques for selecting the target sequence for siRNA are provided, for example, in Tuschl T. et al., “The siRNA User Guide,” revised October 11, 2002, the entire disclosure of which is incorporated herein by reference. Further guidance on target sequence selection and / or siRNA design can be found on the Protocol Online (www.protocol-online.com) website using the keyword “siRNA.” Thus, in some embodiments, the sense strand of the siRNA used in this disclosure comprises a substantially identical nucleotide sequence to any segment of approximately 19 to approximately 25 consecutive nucleotides in the target mRNA.

[0288] siRNA can be obtained using a variety of techniques known to those skilled in the art. For example, siRNA can be chemically synthesized or produced through recombination. Preferably, siRNA is transcribed from a recombinant circular or linear DNA plasmid using any suitable promoter. The selection of other suitable promoters is within the scope of the art. The selection of plasmids suitable for transing siRNA, the method for inserting the nucleic acid sequence for expressing siRNA into the plasmid, and the IVT method for in vitro transcription of said siRNA are all within the scope of the art.

[0289] As used herein, the term “miRNA” refers to a non-coding RNA having a length of 21 to 25 nucleotides (such as 21 to 23, preferably 22) and inducing the degradation of target mRNA and / or preventing the translation of target mRNA. miRNAs are commonly found in plants, animals, and some viruses, where they are encoded by eukaryotic DNA in plants and animals and by viral DNA (in genome-based DNA viruses), respectively. miRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNA), typically leading to translational repression or target degradation and gene silencing.

[0290] miRNAs can be obtained using a variety of techniques known to those skilled in the art. For example, miRNAs 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.). Preferably, miRNAs are transcribed from recombinant circular or linear DNA plasmids using any suitable promoter.

[0291] DNA

[0292] In some embodiments of all aspects of this disclosure, the nucleic acid is DNA. Hereinafter, the term "DNA" refers to a nucleic acid molecule comprising deoxyribonucleotide residues. DNA typically contains naturally occurring nucleic acids such as adenosine (dA), thymidine (dT), cytidine (dC), and guanosine (dG) ("d" stands for "deoxy"). In a preferred embodiment, the DNA contains all or most of the deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide lacking a hydroxyl group at the 2'-position of the β-D-furanose ribosyl group. DNA encompasses, but is not limited to, double-stranded DNA, single-stranded DNA, isolated DNA (such as partially purified DNA), substantially pure DNA, synthetic DNA, recombinant DNA, and modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alteration may refer to the addition of non-nucleotide material to internal DNA nucleotides or to one or more ends of DNA. It is also contemplated herein that the nucleotides in the DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the purposes of this disclosure, such altered DNA is considered an analogue of naturally occurring DNA. A molecule is considered to contain "majority deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is greater than 50% based on the total number of nucleotide residues in the molecule (e.g., 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%). The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (regardless of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or their analogues). DNA can be recombinant DNA and can be obtained by cloning nucleic acids, particularly cDNA. cDNA can be obtained by reverse transcription of RNA. DNA can include plasmids, nanoparticles, microcircles, transposons, or linear DNA such as doggybone DNA.

[0293] Pharmacologically active peptides or polypeptides

[0294] "Encoding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide (such as a gene, cDNA, or RNA (preferably mRNA)) to act as a template for the synthesis of other polymers and macromolecules in biological processes, which have defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences and the resulting biological properties. Thus, if the transcription and translation of RNA (preferably mRNA) corresponding to a gene produces a protein in a cell or other biological system, then the gene encodes that protein. Similarly, if the translation of RNA (e.g., in a cell) produces a protein, then that RNA (such as mRNA) encodes that protein.

[0295] In some embodiments, the active ingredient is RNA (preferably mRNA) or DNA as described in this disclosure, comprising a nucleic acid sequence (e.g., ORF) encoding one or more polypeptides (e.g., peptides or proteins, preferably pharmaceutically active peptides or proteins). In some embodiments, the RNA (preferably mRNA) or DNA described in this disclosure is capable of expressing the peptide or protein, particularly if transferred to cells or a subject. Thus, in some embodiments, the RNA (preferably mRNA) or DNA described in this disclosure contains a coding region (ORF) encoding a peptide or protein, preferably a pharmaceutically active peptide or protein. In this regard, an "open reading frame" or "ORF" is a continuous segment of codons that begins with a start codon and ends with a stop codon. Such RNA (preferably mRNA) or DNA encoding pharmaceutically active peptides or proteins is also referred to herein as "pharmaceutical active RNA" (or "pharmaceutical active mRNA") or "pharmaceutical active DNA". In some embodiments, the RNA (preferably mRNA) or DNA described in this disclosure comprises a nucleic acid sequence encoding more than one peptide or polypeptide (e.g., two, three, four, or more peptides or polypeptides). In some embodiments, the RNA (preferably mRNA) or DNA described in this 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 therapies. In some embodiments, the lipid particle composition comprising RNA or DNA may comprise one or more types of RNA or DNA, wherein each RNA or DNA encodes a different peptide or protein.

[0296] Preferably, the RNA (i) contains structural elements (5' cap, 5' UTR, 3' UTR, polyadenylate sequence) optimized for maximum efficacy in terms of RNA stability and translation efficiency; (ii) is modified to optimize RNA efficacy (e.g., improve translation efficiency, reduce immunogenicity, and / or reduce cytotoxicity) (e.g., by replacing (partially or completely, preferably completely) naturally occurring nucleosides (especially cytidine) with synthetic nucleosides (e.g., modified nucleosides selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine); and / or codon optimization), or (iii) both (i) and (ii).

[0297] The term "pharmacologically active peptide or protein" can be understood to mean a peptide or protein that can be used to treat an individual in whom the expression of the peptide or protein would benefit (e.g., in improving symptoms of a disease or disorder). Preferably, the pharmacologically active peptide or protein has therapeutic or palliative properties and can be administered to improve, alleviate, reduce, reverse, delay the onset of one or more symptoms of a disease or disorder, or reduce their severity. Pharmacologically active peptides or proteins may have preventative properties and can be used to delay the onset of a disease or disorder or reduce the severity of such a disease or disorder.

[0298] Specific examples of pharmacologically active peptides and proteins include, but are not limited to, cytokines, interferons such as interferon-α (IFN-α), interferon-β (IFNβ), or interferon-γ (IFN-γ), 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 include antibodies (e.g., IgG), T-cell receptors (TCR), major histocompatibility complex (MHC) molecules, co-receptors (e.g., CD4, CD8, CD19), antigen receptor helper molecules (e.g., CD-3γ, CD3-δ, CD-3ε, CD79a, CD79b), co-stimulatory or inhibitory molecules (e.g., CD28, CD80, CD86); other immunologically active compounds, such as tumor-associated antigens, pathogen-associated antigens (e.g., bacterial, parasitic, or viral antigens), allergens, and autoantigens.

[0299] In some implementations, the nucleic acid encodes an antigen receptor, such as a T-cell receptor (TCR) or a chimeric antigen receptor (CAR). The pharmaceutically active peptide or protein may be or contains a TCR or CAR. Delivery of the nucleic acid encoding an antigen receptor (such as a TCR or CAR) to cells can be used to generate genetically modified immune effector cells that express the antigen receptor. The functionalized nucleic acid-lipid particles described herein can be used for targeted delivery of nucleic acids encoding antigen receptors, for example, to generate genetically modified in vitro / ex vivo or in vivo immune effector cells that express the antigen receptor. The terms “genetically modified,” “genetically modified,” or simply “modified,” include transfection of cells with nucleic acids. The term “transfection” refers to the introduction of nucleic acids (e.g., DNA and / or RNA) into cells. Cells may be present in a subject (e.g., a patient) or cells may be in vitro (e.g., outside the patient). Transfection can be transient or stable. For example, RNA or DNA can be transfected into cells to transiently express the protein it encodes. Typically, nucleic acids are not integrated into the nuclear genome and will be diluted or degraded by mitosis. Alternatively, transfected nucleic acids typically require stable transfection to enter the cell's genome and remain in its daughter cells. Such stable transfection can be achieved, for example, by using virus-based or transposon-based systems for transfection. Therefore, at least a portion of the transfected DNA can be inserted into the genome for stable transfection. Typically, cells genetically modified to express antigen receptors are stably transfected with nucleic acids encoding the antigen receptors. RNA can be transfected into cells to transiently express its encoded proteins.

[0300] Particle forming components

[0301] The functionalized particles of the present invention also contain a particle-forming component.

[0302] In one embodiment, the one or more particle-forming components comprise cationic lipids or cationic ionizable lipids as defined and exemplified below. In one embodiment, such particles are lipid nanoparticles (LNPs) as defined above. In one embodiment, such particles are lipid complexes (LPXs) as defined above. In such particles, the portion L, as defined below, is typically a hydrophobic portion, such as a lipid.

[0303] In one embodiment, one or more particle-forming components comprise a cationic polymer, as defined and illustrated below. In one embodiment, the particles formed from the cationic polymer are polymer complexes (PLX) as defined above. In such particles, the portion L, as defined below, is typically an anionic polymer as defined and illustrated below.

[0304] In one embodiment, the particles formed from the cationic polymer are esterified polymer complexes (LPLX) as defined above. In such particles, the portion L, as defined below, is typically a hydrophobic portion, such as a lipid.

[0305] Linker compounds

[0306] In one aspect, the functionalized particles described herein also include a linker compound incorporated into the particles by means of a portion of the linker compound incorporated into the particles. The linker compound comprises: (i) a portion capable of incorporating the linker compound into the particles, and (ii) a first interacting portion.

[0307] The portion of the linker compound incorporated into the particle is connected to a binding portion (first interaction portion) for docking compounds as described below. The portion of the linker compound incorporated into the particle refers to the portion of the linker compound integrated into the particle containing a payload (such as a nucleic acid payload). The binding portion of the linker compound refers to the portion of the linker compound that forms a binding pair for docking compounds.

[0308] Typically, the linker compound is non-covalently incorporated into the loaded particle, i.e., it forms part of the particle, and the binding portion of the linker compound is covalently attached to the portion of the particle in which the linker compound is incorporated in such a manner that it can be used to bind with the docking compound.

[0309] In some implementations, the binding portion of the linker compound comprises a peptide or protein (e.g., an antibody or antibody fragment or peptide tag).

[0310] In some embodiments, the binding portion of the linker compound comprises a peptide or protein (e.g., an antibody or antibody fragment or peptide tag) and is chemically linked, for example, by a linker base to a portion of the particle incorporating the linker compound.

[0311] The linker compounds used herein comprise portions incorporating the linker compound into particles, allowing it to be anchored within the particles. Typically, the portion incorporating the linker compound into the particles interacts with the particles (e.g., one or more particle-forming components) through electrostatic interactions (e.g., in polymer complexes) or hydrophobic interactions (e.g., in LNPs). In some embodiments, the portion incorporating the linker compound into the particles comprises a hydrophobic portion, such as a lipid, like a hydrophobic lipid tail. In some embodiments, the linker compound is incorporated into the particles by interacting the hydrophobic portion of the portion incorporating the linker compound into the particles with the hydrophobic portion of the particle-forming component (such as a lipid bilayer or an oily lipid core). In some embodiments, the portion incorporating the linker compound into the particles comprises a charged portion, such as a charged polymer. In some embodiments, the linker compound is incorporated into the particles by interacting the charge in the portion incorporating the linker compound into the particles with the opposite charge in the particles (e.g., particles with opposite charges and / or particles with net opposite charges in the particle-forming component (e.g., considering all charges of the particle-forming component or considering all charges of the particle-forming component and the nucleic acid load)). In some embodiments, the linker compound is incorporated into the particle by interacting the negative charge in the portion of the linker compound incorporated into the particle with the positive charge of the particle.

[0312] In some embodiments, the surface charge of the particles can be adjusted by incorporating a linker compound. In some embodiments, the surface charge can be adjusted based on the amount and type of the linker compound, preferably based on the charged portion of the linker compound. In embodiments, the type and / or length of the charged portion of the linker compound is used to adjust the surface charge.

[0313] In some embodiments, the linker compound is incorporated into particles, such as those containing cationic particle-forming components like cationic polymers, by interacting the negative charge of a portion of the linker compound incorporated into the particles with the positive charge of the particles. In some embodiments, the linker compound comprises a portion incorporated into particles containing anionic polymers.

[0314] In some embodiments, the linker compound comprises a hydrophobic component (e.g., a lipid component) that allows it to be integrated into (or incorporated into or anchored to) particles comprising a particle-forming component (such as a cationic lipid or cationic ionizable lipid, or a cationic polymer comprising a hydrophobic portion) via non-covalent hydrophobic interactions (e.g., with the hydrophobic (e.g., lipid) core of the particle). For example, the linker compound may comprise a hydrophobic group (e.g., a lipid), such as at least one alkyl chain, providing hydrophobic anchoring to the particles (e.g., LNP, LPX, LPLX), as described herein.

[0315] In one embodiment, the linker compound comprises a compound of formula (A) having a portion L as further described herein, the portion L having a binding portion B1 optionally covalently attached thereto via polymer P (when present) and / or linking portions X1 and X2. This may also be referred to herein as a “targeting compound.” In a first aspect of the invention, the linker compound is a compound of formula (A) as defined herein.

[0316] The L portion of the linker compound refers to the portion of the linker compound integrated into the particle containing the load. The B3 binding portion of the linker compound refers to the portion of the linker compound that binds to IgD on the target cell.

[0317] In one implementation, L is the hydrophobic portion.

[0318] In one implementation, L is a lipid.

[0319] In one embodiment, L is an anionic polymer.

[0320] In one embodiment, the linker compound is a compound of formula (A):

[0321] L‐X1‐P‐X2‐B1 (A);

[0322] in:

[0323] P either does not exist or contains a polymer;

[0324] L contains a portion capable of incorporating the compound into the particles, which, when P is absent, is attached to B1, or, when P is present, is attached to the first end of polymer P; and

[0325] X1 and X2 are either independent or connected parts.

[0326] In one implementation, a portion of B1 is selected from the group consisting of antibodies, antibody-like molecules, VHH, or peptides.

[0327] In one implementation, part B1 is VHH.

[0328] In one implementation, part B1 is a peptide.

[0329] Typically, linker compounds are non-covalently incorporated into particles containing active components, i.e., they form part of the particles, and the binding portion of the linker compound is covalently attached to the hydrophobic portion in such a way that it can be used to bind to target cells or docking compounds.

[0330] In some embodiments, the binding portion B1 of the linker compound contains a peptide or protein (e.g., an antibody or antibody fragment or peptide tag).

[0331] In some embodiments, the binding portion B1 of the linker compound comprises a peptide or protein (e.g., an antibody or antibody fragment or peptide tag) and is chemically linked to a hydrophobic portion (e.g., a lipid) via a linker base.

[0332] In one embodiment, the linker compound described herein comprises a hydrophobic component (e.g., a lipid component), which allows it to be anchored within particles. In some embodiments, the hydrophobic component comprises a portion selected from: a vitamin E compound (which may be α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrienol, γ-tocotrienol, δ-tocotrienol, preferably α-tocopherol), a dialkylamine, such as dimyristicamine (DMA), a diacylglycerol ester, such as 1,2-dimyristicoyl-sn-glycerol (DMG), and a ceramide. In some embodiments, the hydrophobic moiety comprises two C8-C24 hydrocarbon chains. In some embodiments, the hydrophobic moiety comprises two C10-C18 hydrocarbon chains.

[0333] In some embodiments, the linker compound described herein has a phospholipid as a hydrophobic group (e.g., lipid), such as a biodegradable phospholipid, like phosphatidylethanolamine. In some embodiments, the linker compound described herein has a glycerophospholipid as a hydrophobic group (e.g., lipid). In some embodiments, the phospholipid is selected from the group consisting of DSPE (distearylphosphatidylethanolamine), DPPE (dispalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleoylphosphatidylethanolamine) and mixtures thereof. In some embodiments, DSPE, as a phospholipid, will be used for its stability properties in the particles described herein. Furthermore, as a hydrophobic group (e.g., lipid), a compound having at least one alkyl chain that provides a hydrophobic anchor to the particles as described herein can be used.

[0334] In some embodiments, the linker compound comprises a polymer as defined herein, which forms part P in its presence. In some embodiments, the hydrophobic portion (e.g., lipid) or anionic polymer of the linker compound and the binding portion of the linker compound are linked (typically covalently) by polymer P.

[0335] In some embodiments, the linker compound is present in an amount of 0.01 mol% to 10 mol%, optionally 0.05 mol% to 5 mol% of the lipid mixture. In some embodiments, the linker compound is present in an amount of 0.1 mol% to 2 mol%, optionally 0.1 mol% to 1 mol% of the lipid mixture.

[0336] In one embodiment of formula (A) or (A'), the hydrophobic portion comprises a lipid. In one embodiment of formula (A) or (A'), the hydrophobic portion comprises a phospholipid. In one embodiment of formula (A) or (A'), the hydrophobic portion comprises a portion selected from the group consisting of DSPE (distearylphosphatidylethanolamine), DPPE (dispalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleoylphosphatidylethanolamine) and mixtures thereof. In one embodiment of formula (A) or (A'), the hydrophobic portion comprises a DSPE portion.

[0337] In one embodiment of formula (A) or (A'), 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 thereof, and combinations thereof.

[0338] In one embodiment of formula (A) or (A'), P comprises polyethylene glycol (PEG), preferably wherein the average molecular weight of PEG is about 200 to about 10,000, more preferably 500 to 5000, even more preferably 1000 to 4000, and most preferably 2000.

[0339] In one embodiment of formula (A) or (A'), P comprises the following general formula:

[0340]

[0341] Where n is between 1 and 100.

[0342] In one embodiment of formula (A) or (A'), X1 comprises a carbonyl group.

[0343] In one embodiment of formula (A) or (A'), X2 comprises a reaction product of a thiol or cysteine ​​reactive group and a thiol or cysteine ​​group of a compound comprising the binding moiety B. In one embodiment of formula (A) or (A'), the thiol or cysteine ​​reactive group comprises a maleimide group.

[0344] In one embodiment of formula (A) or (A'), the hydrophobic portion having the binding portion covalently attached thereto comprises a distearate-glycero-phosphoethanolamine-polyethylene glycol-conjugate (DSPE-PEG).

[0345] In some embodiments, the linker compound is a compound of formula (A1):

[0346] L‐X1‐P‐X2‐B1 (A1);

[0347] in:

[0348] P contains polymers;

[0349] L includes a hydrophobic portion (e.g., lipid) or anionic polymer attached to a first end of polymer P;

[0350] B1 includes a portion capable of binding to B2 of a compound of formula (I) as defined below, wherein portion B1 is attached to the second end of polymer P;

[0351] X1 either does not exist or is the first connection part; and

[0352] X2 either does not exist or is a connection part.

[0353] In some embodiments of formula (A1), X1 contains a carbonyl group.

[0354] In some embodiments of formula (A1), L comprises phosphatidylethanolamine, which can be linked to P via an amide group.

[0355] In some embodiments of formula (A1), X2 comprises a reaction product of a thiol or cysteine ​​reactive group (e.g., a maleimide group) with a thiol or cysteine ​​group of a compound containing a binding moiety.

[0356] In some embodiments of formula (A1), L comprises lipids as described above. In some embodiments of formula (A1), L comprises DSPE (distearylphosphatidylethanolamine), DPPE (dispalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleoylphosphatidylethanolamine), which may be linked to P via an amide group.

[0357] In some embodiments of formula (A1), P comprises a polymer as described above. In some embodiments of formula (A1), P comprises a polymer that provides stealth properties, prolongs cyclic half-life, and / or reduces nonspecific protein binding or cell adhesion.

[0358] In some embodiments of formula (A1), 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 (A1), P comprises poly(ethylene glycol) (PEG); for example, PEG as described above.

[0359] In some embodiments of formula (A1), L-X1-P comprises an amphiphilic derivative of the polymer as described above. In some embodiments of formula (A1), the amphiphilic derivative of the polymer comprises a conjugate of distearyl-glycerol-ethanolamine phosphate (DSPE) and the polymer (e.g., the polymer described above). In some embodiments of formula (A1), the amphiphilic derivative of the polymer comprises a distearyl-glycerol-ethanolamine phosphate-polyethylene glycol-conjugate (DSPE-PEG).

[0360] In some embodiments of formula (A1), the linker compound is obtained by reacting a thiol or cysteine ​​reactive group of a reagent comprising an amphiphilic derivative of a polymer (e.g., a PEG reagent comprising a hydrophobic portion (e.g., a lipid)) with a thiol or cysteine ​​group of a compound comprising the linker moiety. In some embodiments of formula (A1), the thiol or cysteine ​​reactive group comprises a maleimide group.

[0361] In some embodiments of formula (A1), the PEG reagent comprises DSPE-PEG-maleimide. In some embodiments of formula (A1), the compound comprising the binding moiety comprises the formula HS-(CH2). n C(O)-B1, where n ranges from 1 to 5 and B1 contains the binding portion. In some embodiments, n is 2.

[0362] In some embodiments of formula (A1), the linker compound comprises 1,2-distearate-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol)] and includes HS-(CH2). n The reaction product of the compound C(O)-B1, wherein n ranges from 1 to 5 and B1 comprises the binding moiety. In some embodiments of formula (A1), n ​​is 2.

[0363] In some embodiments, the linker compound has the general formula (A2):

[0364] L-X1-P-X2-B1 (A2)

[0365] L, X1, P, and B are as described above, and X2 contains the thiosuccinimide moiety.

[0366] In some embodiments, the linker compound comprises the following general formula (A2').

[0367]

[0368] (A2')

[0369] Wherein B1 contains the binding portion, and PEG is polyethylene glycol as defined above (either in its broadest or preferred aspects).

[0370] In some embodiments of formula (A2) or (A2'), B1 includes a portion comprising the structure -N-peptide-C(O)-NH2, wherein the peptide portion is as defined herein.

[0371] In some embodiments, the linker compound has the following general formula (A3):

[0372] (A3)

[0373] P, X2, and B1 are as described above, and R1 and R2 independently contain alkyl moieties as defined herein (whether in their broadest or preferred aspects).

[0374] In some embodiments, at least one, for example, each alkyl moiety is straight-chain or branched, preferably straight-chain. In some embodiments, at least one, for example, each alkyl moiety has at least 8 carbon atoms, for example, 8 to 24, such as 10 to 18 carbon atoms. Preferably, at least one, for example, each alkyl moiety is an alkyl moiety of a fatty acid alcohol; more preferably, at least one, for example, each alkyl moiety is an alkyl moiety of a fatty acid alcohol having at least 8 carbon atoms, for example, 8 to 24, such as 10 to 18 carbon atoms. Examples of alkyl moiety include -(CH2). 17 CH3 (stearyl group), -(CH2) 15 CH3 (palmitoyl) and -(CH2) 13 CH3 (Myristyl).

[0375] In some embodiments of formula (A3), R1R2N- in formula (A3) above is 1,2-dimyristylamine, wherein both alkyl groups are -(CH2). 13 CH3 (Myristyl).

[0376] In some embodiments of formula (A3), polymer P comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylamino-ethoxy)ethoxy)acetic acid (pMAEEA), or derivatives thereof. In some embodiments of formula (A3), polymer P comprises the following general formula:

[0377]

[0378] Where n is 5 to 50, for example, 5 to 25, for example, 7 to 14, for example, 10 to 25, for example, 14 to 17. In some embodiments of formula (A3), n is 8 or 14. In some embodiments of formula (A3), n is 14. In some embodiments of formula (A3), R1 and R2 in the above formula are -(CH2). 13 CH3 (myristyl) and polymer P contains the following general formula:

[0379]

[0380] Where n is 14.

[0381] In some embodiments, the linker compound has the general formula (A4):

[0382] (A4)

[0383] Where P, X2, and B1 are as described above, and R t1 and R t2 Each of them is independently either H or methyl.

[0384] In some implementations of formula (A4), R t1 and R t2 Both are methyl groups. In some embodiments of formula (A4), R t1 It is methyl, and R t2 It is H. In some implementations of formula (A4), R t1 It is H, and R t2 It is methyl. In some embodiments of formula (A4), R t1 and R t2 Both are H.

[0385] In some embodiments, the linker compound has the general formula (A4'):

[0386] (A4”)

[0387] P, X2, and B1 are as described above.

[0388] In some embodiments of formula (A4) or (A4'), polymer P in the above formula comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)-ethoxy)acetic acid (pMAEEA), or derivatives thereof. In some embodiments of formula (A4) or (A4'), polymer P comprises the following general formula:

[0389]

[0390] Where n is 5 to 50, for example, 5 to 25, for example, 7 to 14, for example, 10 to 25, for example, 14 to 17. In some embodiments of formula (A4), (A4'), (A4”), or (A4'), n is 8 or 14. In some embodiments of formula (A4), (A4'), (A4”), or (A4'), n is 8. In some embodiments of formula (A4), (A4'), (A4”), or (A4'”), n is 14.

[0391] In some embodiments, the linker compound has the general formula (A5):

[0392] (A5)

[0393] X1, P, X2 and B1 are as described above, and R1 and R2 independently contain acyl moieties.

[0394] In some embodiments of formula (A5), at least one, for example, each acyl moiety is linear or branched, preferably linear. In some embodiments of formula (A5), at least one, for example, each acyl moiety has at least 8 carbon atoms, for example, 8 to 24, such as 10 to 18 carbon atoms. Preferably, at least one, for example, each acyl moiety is an acyl moiety of a fatty acid; more preferably, at least one, for example, each acyl moiety is an acyl moiety of a fatty acid having at least 8 carbon atoms, for example, 8 to 24, such as 10 to 18 carbon atoms. Examples of acyl moiety include CH3 (CH2). 16 C(O)-(stearoyl), CH3(CH2) 14 C(O)-(palmitoyl) and CH3(CH2) 12 C(O)-(myristoyl). In some embodiments of formula (A5) or (A5'), both acyl groups are CH3(CH2). 16 C(O)-(stearoyl). In some embodiments of formula (A5) or (A5'), both acyl groups are CH3(CH2). 12 C(O)-(myristoyl). In some embodiments of formula (A5), X1 may or may not contain -HPO3.- (CH2) n -NH-, where n is 1 to 5, for example, 2.

[0395] In some embodiments of formula (A5), polymer P comprises poly-2-(2-(2-aminoethoxy)ethoxy)-acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)-ethoxy)acetic acid (pMAEEA), or derivatives thereof. In some embodiments of formula (A5), polymer P comprises the following general formula:

[0396]

[0397] Where n is 5 to 50, for example, 5 to 25, for example, 7 to 14, for example, 10 to 25, for example, 14 to 17. In some embodiments of formula (A5) or (A5'), n is 8 or 14. In some embodiments of formula (A5) or (A5'), n is 8. In some embodiments, n is 14.

[0398] In some embodiments of formula (A5), polymer P comprises pSar. In some embodiments of formula (A5), polymer P comprises the following general formula:

[0399]

[0400] Where s is 2 to 200, for example, 5 to 100, for example, 10 to 50, for example, 15 to 40. In some embodiments of formula (A5), s is 20 or 23.

[0401] In some embodiments, the linker compound (having a hydrophobic portion of the binding portion covalently attached thereto) comprises the following general formula (A5'):

[0402] (A5')

[0403] P, X2, and B1 are as described above, and R1 and R2 independently contain acyl moieties.

[0404] In some embodiments of formula (A5'), at least one, for example, each acyl moiety is linear or branched, preferably linear. In some embodiments of formula (A5'), at least one, for example, each acyl moiety has at least 8 carbon atoms, for example, 8 to 24, such as 10 to 18 carbon atoms. Preferably, at least one, for example, each acyl moiety is an acyl moiety of a fatty acid; more preferably, at least one, for example, each acyl moiety is an acyl moiety of a fatty acid having at least 8 carbon atoms, for example, 8 to 24, such as 10 to 18 carbon atoms. Examples of acyl moiety include CH3 (CH2). 16C(O)-(stearoyl), CH3(CH2) 14 C(O)-(palmitoyl) and CH3(CH2) 12 C(O)-(myristoyl). In some embodiments of formula (A5'), both acyl groups are CH3(CH2). 16 C(O)-(stearoyl). In some embodiments of formula (A5'), both acyl groups are CH3(CH2). 12 C(O)-(myristoyl). In some embodiments of formula (A5'), polymer P comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)-ethoxy)acetic acid (pMAEEA), or derivatives thereof. In some embodiments of formula (A5'), polymer P comprises the following general formula:

[0405]

[0406] Where n is 5 to 50, for example, 5 to 25, for example, 7 to 14, for example, 10 to 25, for example, 14 to 17. In some embodiments of formula (A5'), n is 8 or 14. In some embodiments of formula (A5'), n is 8. In some embodiments of formula (A5'), n is 14.

[0407] In some implementations of formula (A5'), n is 8 and R1 and R2 are CH3(CH2). 16 C(O)-(stearoyl). In some embodiments of formula (A5'), n is 14 and R1 and R2 are CH3(CH2). 16 C(O)-(stearoyl). In some embodiments of formula (A5'), n is 8 and R1 and R2 are CH3(CH2). 12 C(O)-(myristoyl). In some embodiments of formula (A5'), n is 14 and R1 and R2 are CH3(CH2). 12 C(O)-(Myristoyl).

[0408] In some embodiments of formula (A5'), polymer P comprises pSar. In some embodiments of formula (A5'), polymer P comprises the following general formula:

[0409]

[0410] Where s is 2 to 200, for example, 5 to 100, for example, 10 to 50, for example, 15 to 40. In some embodiments, s is 20 or 23. In some embodiments, s is 20 and R1 and R2 are CH3(CH2). 16C(O)-(stearoyl). In some embodiments, s is 20 and R1 and R2 are CH3(CH2). 12 C(O)-(Myristoyl).

[0411] In some embodiments of formulas (A), (A1), (A2), (A2'), (A3), (A4), (A4'), (A5), or (A5') above, X2 comprises a reaction product of a thiol or cysteine ​​reactive group (e.g., a maleimide group) with a compound comprising a thiol or cysteine ​​group. In some embodiments of formulas (A), (A1), (A2), (A2'), (A3), (A4), (A4'), (A5), or (A5') above, the compound comprising a thiol or cysteine ​​group comprises the formula HS(CH2)nC(O)-, where n ranges from 1 to 5. In some embodiments, n is 2. In some embodiments, X2 comprises a thiosuccinimide moiety.

[0412] In some implementations, X2 includes the following general formula:

[0413] .

[0414] In some embodiments of the above formulas (A), (A1), (A2), (A2'), (A3), (A4), (A4'), (A5), or (A5'), X2 comprises the following general formula:

[0415]

[0416] Where n1 and n2 are independently 1 to 5. In some embodiments, n1 is 1 and n2 is 2. In some embodiments, n1 is 2 and n2 is 1.

[0417] In some embodiments of linker compounds such as (A), (A1), (A2), (A2'), (A3), (A4), (A4'), (A5) or (A5') above, the binding portion B1 includes an epitope tag, such as an ALFA-tag, as described herein.

[0418] In one embodiment, the linker compound has the general formula (A10):

[0419] L-X1-P-X2-B1 (A10)

[0420] in:

[0421] P contains polymers;

[0422] L includes a hydrophobic portion (e.g., a lipid) or an anionic polymer attached to a first end of the polymer;

[0423] B1 contains an epitope tag attached to the second end of the polymer, such as an ALFA-tag, as described herein;

[0424] X1 either does not exist or is the first connection part; and

[0425] X2 either does not exist or is a second connection part.

[0426] In some embodiments of formula (A10), X1 comprises a carbonyl group. In some embodiments of formula (A10), L comprises phosphatidyl-ethanolamine, which can be linked to P via an amide group.

[0427] In some embodiments of formula (A10), X2 comprises a reaction product of a thiol or cysteine ​​reactive group (e.g., a maleimide group) with a thiol or cysteine ​​group of a compound containing an epitope tag. In some embodiments of formula (A10) or A10a, X2 comprises a thiosuccinimide moiety.

[0428] In some embodiments of formula (A10), L comprises lipids as described above. In some embodiments of formula (A10), L comprises DSPE (distearylphosphatidylethanolamine), DPPE (dispalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleoylphosphatidylethanolamine), which may be linked to P via an amide group.

[0429] In some embodiments of formula (A10), P comprises a polymer as described above. In some embodiments of formula (A10), P comprises a polymer that provides stealth properties, prolongs cyclic half-life, and / or reduces nonspecific protein binding or cell adhesion. In some embodiments of formula (A10), 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). In some embodiments of formula (A10), P comprises polyethylene glycol (PEG); for example, PEG as described above.

[0430] In some embodiments of formula (A10), L-X1-P comprises an amphiphilic derivative of the polymer as described above. In some embodiments of formula (A10), the amphiphilic derivative of the polymer comprises a conjugate of distearyl-glycerol-ethanolamine phosphate (DSPE) and the polymer (e.g., the polymer described above). In some embodiments of formula (A10), the amphiphilic derivative of the polymer comprises a distearyl-glycerol-ethanolamine phosphate-polyethylene glycol-conjugate (DSPE-PEG).

[0431] In some embodiments of formula (A10), the linker compound is obtained by reacting a 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., a lipid)) with a thiol or cysteine ​​group of a compound comprising a major targeting moiety or epitope tag. In some embodiments of formula (A10), the thiol or cysteine ​​reactive group comprises a maleimide group. In some embodiments of formula (A10), the PEG reagent comprises DSPE-PEG-maleimide. In some embodiments of formula (A10), the compound comprising a major targeting moiety or epitope tag comprises the formula HS(CH2). n C(O)-B, where n ranges from 1 to 5 and B contains a primary target portion (i.e., the portion capable of binding to IgD) or an epitope tag. In some embodiments of formula (A10), n is 2.

[0432] In some embodiments of formula (A10), the linker compound comprises 1,2-distearate-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol)] and includes the formula HS(CH2). n The reaction product of a compound of C(O)-B, wherein n ranges from 1 to 5 and B contains a major targeting moiety (i.e., the moiety capable of binding to IgD) or an epitope tag. In some embodiments of formula (A10), n is 2.

[0433] In some embodiments, the linker compound has the following general formula (A10b):

[0434] (A10b)

[0435] B1 contains epitope tags, such as ALFA-tags, as described herein.

[0436] In some embodiments, the linker compound has the following general formula (A10d).

[0437] (A10d)

[0438] X2 is as described above, and R1 and R2 are CH3(CH2). 16 C(O)-(stearoyl) or CH3(CH2) 12 C(O)-(myristoyl), polymer P contains the following general formula:

[0439]

[0440] Where n is 5 to 50, for example, 5 to 25, for example, 7 to 14, for example, 10 to 25, for example, 14 to 17, for example, 8 or 14, and B1 contains epitope labels, for example, ALFA-labels, such as the ALFA-labels described herein.

[0441] In some implementations of formula (A10d), n is 8 and R1 and R2 are CH3(CH2). 16 C(O)-(stearoyl). In some embodiments of formula (A10d), n is 14 and R1 and R2 are CH3(CH2). 16 C(O)-(stearoyl). In some embodiments of formula A10d) or (A10e), n is 8 and R1 and R2 are CH3(CH2). 12 C(O)-(myristoyl). In some embodiments of formula (A10d), n is 14 and R1 and R2 are CH3(CH2). 12 C(O)-(myristoyl). In some embodiments of formula (A10d), X2 has the following general formula:

[0442] .

[0443] In some embodiments, the linker compound comprises the following general formula (A10f):

[0444] (A10f)

[0445] X2 is as described above, and R1 and R2 are CH3(CH2). 16 C(O)-(stearoyl) or CH3(CH2) 12 C(O)-(myristoyl), polymer P contains the following general formula:

[0446]

[0447] Where s is 2 to 200, for example, 5 to 100, for example, 10 to 50, for example, 15 to 40, for example, 20 or 23, and

[0448] B1 contains epitope tags, such as ALFA-tags, as described herein.

[0449] In some embodiments of formula (A10f), s is 20 and R1 and R2 are CH3(CH2). 16 C(O)-(stearoyl). In some embodiments of formula (A10f), s is 20 and R1 and R2 are CH3(CH2). 12 C(O)-(Myristoyl).

[0450] In some embodiments of formula (A10f), X2 includes the following general formula:

[0451] .

[0452] In some embodiments of formula (A10f), B1 includes a portion comprising the structure -N-peptide-C(O)-NH2, wherein the peptide contains an epitope tag, such as an ALFA-tag, as described herein.

[0453] In one aspect, this disclosure provides linker compounds as described above, which are integrated into particles (e.g., particles as described herein) via a hydrophobic component (e.g., a lipid component) of the linker compound.

[0454] In one embodiment of the linker compound, the binding portion B1 comprises a peptide or a polypeptide.

[0455] In one embodiment of the linker compound, the peptide tag comprises an ALFA-tag.

[0456] In some embodiments, the linker compound has the formula (A20):

[0457] L‐X1‐P‐X2‐B1 (A20)

[0458] in

[0459] P contains polymers;

[0460] L includes a hydrophobic portion or an anionic polymer attached to the first end of the polymer;

[0461] B includes a bonding portion attached to the second end of the polymer;

[0462] X1 either does not exist or is the first connection part; and

[0463] X2 either does not exist or is a second connection part.

[0464] In some embodiments of formula (A20), X1 contains a carbonyl group.

[0465] In some embodiments of formula (A20), X2 comprises the reaction product of a maleimide group and a thiol or cysteine ​​group of a compound containing the binding moiety.

[0466] In some embodiments of formula (A20), the hydrophobic portion is a lipid or is contained in a lipid. In some embodiments of formula (A20), the lipid comprises a phospholipid, for example, 1,2-distearate-sn-glycero-3-phosphoethanolamine (DSPE).

[0467] In some embodiments of formula (A20), the polymer provides stealth properties, extended cyclic half-life, and / or reduced nonspecific protein binding or cell adhesion.

[0468] In some embodiments of formula (A20), the polymer comprises polyethylene glycol (PEG). The average molecular weight of PEG can range from 200 to 10,000, preferably from 500 to 5,000, more preferably from 1,000 to 4,000, and most preferably from 2,000.

[0469] In some embodiments of formula (A20), the hydrophobic portion having a binding portion covalently attached thereto comprises a distearate-glycero-phosphoethanolamine-polyethylene glycol-conjugate (DSPE-PEG).

[0470] In some embodiments of formula (A20), the binding portion covalently attached to the hydrophobic portion comprises a peptide, and preferably the binding portion comprises an ALFA-tag as defined in more detail below.

[0471] In some embodiments of the linker compounds such as any of the above formulas (A), (A1), (A2), (A2'), (A3), (A4), (A4'), (A5), (A5'), (A10), (A10b), (A10d), (A10f), or (A20), X1 and X2 are covalently linked via an amide, disulfide, thioether, ether, ester, thioester, thioamide, alkylene, alkenylene, alkyneylene, and / or 1,2,3-triazole.

[0472] In some embodiments of the linker compounds such as any of the above formulas (A), (A1), (A2), (A2'), (A3), (A4), (A4'), (A5), (A5'), (A10), (A10b), (A10d), (A10f), or (A20), the cyclized amino acid sequence described herein is generated by linking the amino group of the side chain of one of X1 and X2 to the carboxyl group of the side chain of the other of X1 and X2 via an amide bond. The amino group of the side chain of an amino acid having a side chain 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 the cyclized amino acid sequence via an amide bond.

[0473] In some embodiments of the linker compounds such as any of the formulas (A), (A1), (A2), (A2'), (A3), (A4), (A4'), (A5), (A5'), (A10), (A10b), (A10d), (A10f), or (A20) above, the cyclized amino acid sequence described herein is generated by linking a thiol group of the side chain of one of X1 and X2 to a thiol group of the side chain of the other of X1 and X2 via a disulfide bond. Thiol-containing amino acids include cysteine ​​and other thiol-containing amino acids, such as Pen.

[0474] In some embodiments of the linker compound such as any of the above formulas (A), (A1), (A2), (A2'), (A3), (A4), (A4'), (A5), (A5'), (A10), (A10b), (A10d), (A10f), or (A20), X1 and X2 are independently selected from the group consisting of Glu, DGlu, Asp, DAsp, Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, DDap, Cys, DCys, hCys, DhCys, Pen, and DPen, provided that when When X1 is Glu, DGlu, Asp, or DAsp, X2 is Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, or DDap; when X1 is Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, or DDap, X2 is Glu, DGlu, Asp, or DAsp; and when X1 is Cys, DCys, hCys, DhCys, Pen, or DPen, X2 is Cys, DCys, hCys, DhCys, Pen, or DPen.

[0475] In some embodiments of the linker compound such as any of the above formulas (A), (A1), (A2), (A2'), (A3), (A4), (A4'), (A5), (A5'), (A10), (A10b), (A10d), (A10f), or (A20), X1 is Glu and X2 is Lys. In some embodiments, -cyclo(Glu----Lys)-, -c(Glu----Lys)-, -cyclo(E-----K)-, -c(E------K)-, -E----K-cyclo, or -cycloE---cycloK comprises the following structure:

[0476]

[0477] In some embodiments of the linker compound such as any of the above formulas (A), (A1), (A2), (A2'), (A3), (A4), (A4'), (A5), (A5'), (A10), (A10b), (A10d), (A10f), or (A20), X1 is Lys and X2 is Glu. In some embodiments of the linker compound such as any of the above formulas (A), (A1), (A2), (A2'), (A3), (A4), (A4'), (A5), (A5'), (A10), (A10b), (A10d), (A10f), or (A20), -cyclo(Lys---Glu)-, -c(Lys----Glu)-, -cyclo(K----E)-, -c(K-----E)-, -K----E-cyclo, or cycloK----cycloE comprises the following structure:

[0478]

[0479] In some embodiments of the linker compound such as any of the above formulas (A), (A1), (A2), (A2'), (A3), (A4), (A4'), (A5), (A5'), (A10), (A10b), (A10d), (A10f), or (A20), X1 is Cys and X2 is Cys. In some embodiments of the linker compound such as any of the above formulas (A), (A1), (A2), (A2'), (A3), (A4), (A4'), (A5), (A5'), (A10), (A10'), (A10”), (A10”'), or (A20), -cyclo(Cys---Cys)-, c(Cys---Cys)-, -cyclo(C----C)-, -c(C-----C)-, -C---C-cyclo, or -cycloC----cycloC comprises the following structure:

[0480] In some embodiments of the linker compound such as any of the above formulas (A), (A1), (A2), (A2'), (A3), (A4), (A4'), (A5), (A5'), (A10), (A10b), (A10d), (A10f), or (A20), the cyclic amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 149). In some other embodiments of the linker compound such as any of the above formulas (A), (A1), (A2), (A2'), (A3), (A4), (A4'), (A5), (A5'), (A10), (A10b), (A10d), (A10f), or (A20), the cyclic amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 150). In some other embodiments of the linker compound such as any of the above formulas (A), (A1), (A2), (A2'), (A3), (A4), (A4'), (A5), (A5'), (A10), (A10b), (A10d), (A10f), or (A20), the cyclic amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 151). In some other embodiments of the linker compound such as any of the above formulas (A), (A1), (A2), (A2'), (A3), (A4), (A4'), (A5), (A5'), (A10), (A10b), (A10d), (A10f), or (A20), the cyclic amino acid sequence is -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Glu)- (SEQ ID NO: 152).

[0481] Cyclic peptides can have different cyclic bridging portions that form a ring structure. Preferably, the ring structure includes a chemically stable bridging portion, such as, for example, an amide group, lactone group, ether group, thioether group, disulfide group, alkylene group, alkenyl group, or 1,2,3-triazole. The following are examples illustrating the variability of the bridging portion in a peptide:

[0482]

[0483]

[0484] In one embodiment, when the nucleic acid-lipid particles contain a linker compound such as any of the formulas (A), (A1), (A2), (A2'), (A3), (A4), (A4'), (A5), (A5'), (A10), (A10b), (A10d), (A10f), or (A20) above, such as peptide-conjugated lipids, this allows for the functionalization of the nucleic acid-lipid particles. For example, a binding moiety B1 that specifically binds to the peptide of a peptide-conjugated lipid can bind to the nucleic acid-lipid particle, wherein the binding moiety can also bind to target cells (e.g., by specifically binding to IgD). This can provide targeted delivery of nucleic acids contained within the functionalized nucleic acid-lipid particles. The binding moiety that specifically binds to the peptide of the compound can be an ALFA-tag binding moiety.

[0485] In some embodiments of the linker compound such as any of formulas (A), (A1), (A2), (A2'), (A3), (A4), (A4'), (A5), (A5'), (A10), (A10b), (A10d), (A10f), or (A20) above, the ALFA-tag-binding portion comprises an antibody or antibody fragment, for example, a Camelidae VHH domain. In some embodiments of the linker compound such as any of formulas (A), (A1), (A2), (A2'), (A3), (A4), (A4'), (A5), (A5'), (A10), (A10b), (A10d), (A10f), or (A20) above, the ALFA-tag-binding portion comprises a single-domain antibody (sdAb) or an NbALFA-nanobody. In some embodiments of the linker compound such as any of the above formulas (A), (A1), (A2), (A2'), (A3), (A4), (A4'), (A5), (A5'), (A10), (A10b), (A10d), (A10f), or (A20), the ALFA-tag binding portion comprises a single-domain antibody, for example, a Camelidae VHH domain comprising the CDR1 sequence VTX1SALNAMAMG (SEQ ID NO: 145) (where X1 is I or V), the CDR2 sequence AVSX2RGNAM (SEQ ID NO: 146) (where X2 is E, H, N, D, or S), and the CDR3 sequence LEDRVDSFHDY (SEQ ID NO: 147).

[0486] In some embodiments of the linker compound such as any one of the above formulas (A), (A1), (A2), (A2'), (A3), (A4), (A4'), (A5), (A5'), (A10), (A10b), (A10d), (A10f), or (A20), the ALFA-tag binding portion comprises a single-domain antibody, for example, a camelid VHH domain comprising the CDR1 sequence GVTX1SALNAMAMG (SEQ ID NO: 148) (where X1 is I or V), the CDR2 sequence AVSX2RGNAM (where X2 is E, H, N, D, or S), and the CDR3 sequence LEDRVDSFHDY.

[0487] In some embodiments of the linker compound such as any of the above formulas (A), (A1), (A2), (A2'), (A3), (A4), (A4'), (A5), (A5'), (A10), (A10b), (A10d), (A10f), or (A20), the ALFA-tag binding portion comprises a single-domain antibody, for example, a Camelidae VHH domain.

[0488] The ALFA-tag binding region can contain a single-domain antibody, such as a Camelidae VHH domain containing the following:

[0489] (i) CDRs containing the following sequences:

[0490] CDR1 – GVTISALNAMAMG (SEQ ID NO: 49)

[0491] CDR2 – AVSERGNTY (SEQ ID NO: 53)

[0492] CDR3 – LEDRVDSFHDY (SEQ ID NO: 51); or

[0493] (ii) CDRs containing the following sequences:

[0494] CDR1 – GVTISALNAMAMG (SEQ ID NO: 49)

[0495] CDR2 – AVSERGNAM (SEQ ID NO: 50)

[0496] CDR3 – LEDRVDSFHDY (SEQ ID NO: 51); or

[0497] (iii) CDRs containing the following sequences:

[0498] CDR1 – GVTISALNAMAMG (SEQ ID NO: 49)

[0499] CDR2 – AVSSRGNAM (SEQ ID NO: 52)

[0500] CDR3 – LEDRVDSFHDY (SEQ ID NO: 51);

[0501] Optionally, one or more of the CDRs contain one, two, or three amino acid mutations. In such an implementation, the CDR is provided according to the definition of AbM used by Oxford Molecular's AbM antibody modeling software (see, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (edited by Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg)).

[0502] The CDR for the ALFA-tag binding portion that can specifically bind to the ALFA tag can also be provided based on IMGT or Kabat annotations.

[0503] Appropriately, according to the Kabat notes, the ALFA-label combination may include:

[0504] (i) CDRs containing the following sequences:

[0505] CDR1-ALNAMAMG (SEQ ID NO: 137);

[0506] CDR2-AVSERGNTYYRDSVKG (SEQ ID NO: 138);

[0507] CDR3 – LEDRVDSFHDY (SEQ ID NO: 139); or

[0508] (ii) CDRs containing the following sequences:

[0509] CDR1-ALNAMAMG (SEQ ID NO: 137);

[0510] CDR2-AVSERGNAMYRESVQG (SEQ ID NO: 140);

[0511] CDR3 – LEDRVDSFHDY (SEQ ID NO: 139);

[0512] Optionally, one or more of the CDRs may contain one, two, or three amino acid mutations.

[0513] Appropriately, according to the IMGT annotation, the ALFA-label combination may include:

[0514] (i) CDRs containing the following sequences:

[0515] CDR1-GVTISALNAMA (SEQ ID NO: 141);

[0516] CDR2-VSERGNT (SEQ ID NO: 142);

[0517] CDR3 – HVLEDRVDSFHDY (SEQ ID NO: 143); or

[0518] (ii) CDRs containing the following sequences:

[0519] CDR1-GVTISALNAMA (SEQ ID NO: 141);

[0520] CDR2 – VSERGNA (SEQ ID NO: 144);

[0521] CDR3 – HVLEDRVDSFHDY (SEQ ID NO: 143);

[0522] Optionally, one or more of the CDRs may contain one, two, or three amino acid mutations.

[0523] The ALFA-tag binding portion may contain a CDR consisting of the sequences provided above. The ALFA-tag binding portion may contain a CDR of the VHH sequence of any one of SEQ ID NO: 54-56, regardless of how the CDR is annotated.

[0524] It should be understood that the ALFA-tag binding portion of one, two, or all three of the mutations introduced into the CDR sequence can still bind specifically to the ALFA tag.

[0525] Suitablely, the ALFA-tag binding portion may comprise, or consist of, any of the VHH sequences of SEQ ID NO: 54-56 or variants thereof having at least 80% identity with them.

[0526] Suitablely, the ALFA-tag binding portion may comprise, or consist of, the VHH sequence of SEQ ID NO: 54 or a variant thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity. Suitablely, the VHH may comprise the sequence shown in SEQ ID NO: 54.

[0527] Suitably, the ALFA-tag binding portion may comprise, or consist of, the VHH sequence of SEQ ID NO: 55 or a variant thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity. Suitably, the VHH may comprise the sequence shown in SEQ ID NO: 55.

[0528] Suitablely, the ALFA-tag binding portion may comprise, or consist of, the VHH sequence of SEQ ID NO: 56 or a variant thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity. Suitablely, the VHH may comprise the sequence shown in SEQ ID NO: 56.

[0529] SEQ ID NO: 54

[0530] EVQLVESGGGLVQPGGSLRLSCAASGVTISALNAMAMGWYRQAPGKRREMVAAVSERGNTYYRDSVKGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS

[0531] SEQ ID NO: 55

[0532] EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS

[0533] SEQ ID NO: 56

[0534] EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGEERVMVAAVSSRGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS

[0535] In some embodiments of the linker compound such as any of the formulas (A), (A1), (A1'), (A2), (A2'), (A2”), (A2”'), (A3), (A3'), (A4), (A4'), (A4”), (A4”'), (A5), (A5'), (A5”), (A5”'), (A10), (A10a), (A10b), (A10c), (A10d), (A10e), (A10f), (A10g), (A20), or (A20”), The ALFA-tag binding portion comprises a bispecific antibody targeting both the ALFA-tag and a cell surface antigen. In some embodiments of the linker compound such as any of the above formulas (A), (A1), (A1'), (A2), (A2'), (A3), (A4), (A4'), (A5), (A5'), (A10), (A10b), (A10d), (A10f), or (A20), the ALFA-tag binding portion comprises a portion binding to the peptide containing the ALFA-tag and a portion targeting the cell surface antigen.

[0536] Preferably, the binding moiety is a peptide, and the composition may also contain, as described herein, peptide-conjugated lipids. In this specification, the term "peptide-conjugated lipid" in its broadest sense refers to a lipid or lipid-like material conjugated to a peptide as defined above (whether in the broadest or preferred sense). In this respect, "peptide" is synonymous with "polypeptide" and "protein." In one embodiment, the peptide comprises an ALFA-tag (i.e., the peptide-conjugated lipid may be an ALFA-conjugated lipid). Such peptide-conjugated lipids are described in more detail in WO2023 / 148276.

[0537] In the direct binding implementation, the functionalized particle comprises: (a) one or more particle-forming components; (b) a payload, such as a nucleic acid payload; and (c) a compound comprising: (i) a portion capable of incorporating a linker compound into the particle; and (ii) a portion capable of binding immunoglobulin D (IgD). No linker compound is present because portion B' of the direct binding compound (c) forms the primary targeting portion as described below.

[0538] In this embodiment, the compound (c) directly bound is preferably a compound of formula (A'), as defined above. In some embodiments, the compound of formula (A') is preferably a compound of formula (A), (A1), (A2), (A2'), (A3), (A4), (A4'), (A5), (A5'), (A10), (A10b), (A10d), (A10f), or (A20), wherein a portion of B1 is replaced by a portion of B'. In these embodiments, the portion of B' may take any preferred meaning given for B1 in formulas (A1), (A1'), (A2), (A2'), (A3), (A3'), (A4), (A4'), (A5), (A5'), (A10), (A10b), (A10d), (A10f), or (A20).

[0539] Main target part

[0540] According to this disclosure, a payload (such as a nucleic acid) is specifically delivered to a target cell by providing a portion that binds to immunoglobulin D (IgD) (e.g., IgD antigen on the target cell), thereby targeting the payload-containing particle to a target cell that expresses IgD on its cell surface.

[0541] Therefore, the primary targeting portion according to the invention is the portion capable of binding with IgD. In one aspect of the invention, specifically in a direct binding embodiment, the primary targeting portion is portion B' and forms part of a direct binding compound (e.g., a compound of formula (A')). In another aspect of the invention, the primary targeting portion is portion B3 and forms part of a compound of formula (I).

[0542] Immunoglobulin D (IgD) is an antibody isotype that constitutes approximately 1% of the proteins in the plasma membrane of immature B lymphocytes (of which it is usually co-expressed with IgM). IgD is also produced in a secretory form, present in very small amounts in serum, accounting for 0.25% of serum immunoglobulins. The relative molecular mass and half-life of secretory IgD are 185 kDa and 2.8 days, respectively. Secretory IgD is produced as a monomeric antibody with two delta-type heavy chains and two Ig light chains.

[0543] In some embodiments, specifically direct binding embodiments, the targeting portion that binds to IgD on the target cell is composed of a compound (i.e., a direct binding compound, such as a compound of formula (A') as defined above) that is part of the payload-carrying particle. In these embodiments, the direct binding compound comprises a binding portion B' that binds to IgD on the target cell. In such embodiments of the direct binding compound, the binding portion B' comprises a portion that binds to IgD on the target cell (e.g., a portion that binds to cell surface IgD on the target cell).

[0544] In some embodiments, the portion binding to IgD on the target cell comprises a portion that further includes a compound (i.e., a linker compound, such as the compound of formula (A)) that is part of the payload-carrying particle and a compound (i.e., a docking compound, such as the compound of formula (I)) that includes a portion for binding to the docking compound. In these embodiments, the linker compound itself preferably does not contain a portion binding to IgD on the target cell. Instead, the linker compound contains a binding portion that forms a binding pair of the docking compound (which itself binds to IgD on the target cell). In such embodiments, the binding portion B1 of the linker compound is preferably a peptide tag or a portion bound to a peptide tag; and the portion B2 of the docking compound contains a portion bound to B1 (a portion bound to a peptide tag, or a peptide tag, respectively), and the portion B3 of the docking compound contains a portion capable of binding to IgD.

[0545] IgD on target cells is also referred to as the "primary target" in this paper.

[0546] As used herein, "primary targeting moiety" or "IgD-binding moiety" refers to the portion of a linker compound or docking compound that binds to IgD (e.g., IgD on the surface of the target cell) (e.g., B3 of a compound of formula (I); or B' of a compound of formula (A'), wherein B' or B3 is a portion capable of binding to IgD on the target cell). The IgD-binding moiety can be any peptide or protein capable of binding to IgD (e.g., an antibody or antibody fragment). Specific embodiments of suitable primary targeting moieties used herein include IgD-binding moieties such as antibodies, antibody fragments, and DARPin.

[0547] The primary target portion preferably binds with high specificity and / or high affinity, and the binding to the primary target is preferably stable in vivo.

[0548] Many binding domains are known in the art, including those based on antigen-binding sites of antibodies, antibody mimics, and T-cell receptors. For example, the primary targeting portion may include: single-chain variable fragments (scFv); single-domain binders (e.g., VHH); artificial binders such as DARPin; or single chains derived from T-cell receptors. Antibodies can be full-length antibodies, single-chain antibody fragments, F(ab) fragments, F(ab')2 fragments, F(ab') fragments, single-domain antibodies (sdAb), nanobodies, affinity molecules, fibronectin artificial antibody backbones, anticalin, affilin, DARPin, VHH, VNAR, iBody, affimer, fynomer, domain antibodies (DAb), abdurin / nanobodies, centyrin, alphabet, or nanofibrin.

[0549] The ability of the primary target moiety to bind IgD can be assessed by determining binding affinity. Quantitative assessment or measurement of binding affinity (e.g., establishing KD values) can be determined or measured using methods known in the art, such as by surface plasmon resonance, for example, by using the Biacore® system. In addition to the equilibrium dissociation constant (KD), the association rate constant (Ka(1 / Ms)) and the dissociation rate constant (KD(1 / s)) can also be determined.

[0550] Surface plasmon resonance (SPR) experiments can be performed using, for example, the Biacore T200.

[0551] Methods for determining binding specificity include, but are not limited to, ELISA, Western blotting, immunohistochemistry, flow cytometry, Förster resonance energy transfer (FRET), phage display libraries, yeast double-hybrid screening, immunoprecipitation, bimolecular fluorescence complementation, and tandem affinity purification. Binding affinity can also be determined using methods such as fluorescence quenching and isothermal titration calorimetry.

[0552] Appropriately, the primary target region can be able to bind specifically to the IgD Fc region.

[0553] The illustrative protein sequences of human and rhesus monkey IgD Fc peptides are shown below as SEQ ID NO: 506-509.

[0554] SEQ ID NO: 506 (Human IgD-Fc with a Linker Region)

[0555] GRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPRSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYLAMTPLIPQSKDENSDDYTTFDDVGS

[0556] SEQ ID NO: 507 (human IgD-Fc)

[0557] GRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPRSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK

[0558] SEQ ID NO: 508 (Rhesus monkey IgD-Fc with a linker region)

[0559] GRREKEDEEEKEQQEGETKTPECPSHTQPLGVYLLPPALQDLWFQDKVTFTCFVVGSDLQDAHLSWEVAGKVPKGGMEEGPLEQHSNGSQSQHSRLALPRSLWNAGTSVTCTLNHPSLPSQKLMALREPAAQAPVRLSLNLLASSDPPEAASWLLCEVSDFSPPNILLMWLENQREVNTSWFATTHPTPQPGSTMFWAWSVLRVPGPTSPQPATYTCVVSHEDSRTLLNASRSLEVSYLATTPPIPQSKDENSDDYTTLDDMGS

[0560] SEQ ID NO: 509 (Rhesus monkey IgD-Fc)

[0561] GRREKEDEEEKEQQEGETKTPECPSHTQPLGVYLLPPALQDLWFQDKVTFTCFVVGSDLQDAHLSWEVAGKVPKGGMEEGPLEQHSNGSQSQHSRLALPRSLWNAGTSVTCTLNHPSL PSQKLMALREPAAQAPVRLSLNLLASSDPPEAASWLLCEVSDFSPPNILLMWLENQREVNTSWFATTHPTPQPGSTMFWAWSVLRVPGPTSPQPATYTCVVSHEDSRTLLNASRSLEVS

[0562] Suitably, the major targeting portion can specifically bind to one or more of the following: SEQ ID NO:506-509; or variants having at least 80% sequence identity with them. Suitably, the major targeting portion can bind to SEQ ID NO:506 or variants having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with it. Suitably, the major targeting portion can bind to SEQ ID NO:507 or variants having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with it. Suitably, the major targeting portion can bind to SEQ ID NO:508 or variants having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with it. Suitably, the major targeting portion can bind to SEQ ID NO:509 or variants having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with it. Suitably, the main targeting portion is capable of binding to each of SEQ ID NO: 506 and 508. Suitably, the main targeting portion is capable of binding to each of SEQ ID NO: 506-509.

[0563] Suitablely, the main target region that can bind to IgD can be a single-domain antibody (sdAb).

[0564] In another aspect, the present invention provides sdAbs capable of binding to IgD. The sdAbs can selectively bind to IgD.

[0565] Suitablely, an sdAb capable of binding IgD may contain any feature as described herein that is capable of binding the main target portion of IgD, wherein the main target portion is the sdAb.

[0566] sdAbs (i.e., nanobodies) can be defined as antibody fragments composed of a single monomeric variable antibody domain. An sdAb can be a single-chain variable domain, which can be a heavy chain variable (VH) domain or a light chain variable (VL) domain, and has three CDRs.

[0567] sdAb has been engineered from heavy chain antibodies found in camelids to produce antibodies with variable heavy chain domains (VHH). sdAb has also been engineered from heavy chain antibodies known as immunoglobulin neoantigen receptors (IgNARs) found in cartilaginous fishes to produce antibodies with variable neoantigen receptors (VNARs).

[0568] Suitablely, the sdAb can be a variable heavy chain domain antibody (VHH), a heavy chain variable (VH) domain antibody, or a variable neoantigen receptor antibody (VNAR). The sdAb can be Humabody® (Crescendo Biologics). Humabody is an antibody produced by transgenic mice that generate heavy chain-only antibodies with a fully human VH domain but lacking a VL domain.

[0569] Preferably, sdAb can be VHH.

[0570] The primary target fraction, preferred sdAb, and most preferred VHH can be non-human, humanized, or fully human. Suitably, the primary target fraction sdAb or VHH can be a humanized sdAb. Suitably, the primary target fraction sdAb or VHH can be a fully human primary target fraction sdAb or VHH.

[0571] The remainder of the polypeptide can be any sequence that provides a suitable backbone for the antigen-binding site and is displayed in an appropriate manner to enable it to bind to the antigen.

[0572] Therefore, in one aspect, an sdAb capable of binding to an ALFA-tagged peptide is provided, the sdAb containing a CDR containing the following sequence:

[0573] CDR1 – ALNAMAMG (SEQ ID NO: 137)

[0574] CDR2 – AVSERGNTYYRDSVKG (SEQ ID NO: 138); and

[0575] CDR3 – LEDRVDSFHDY (SEQ ID NO: 139).

[0576] In one implementation, sdAb comprises SEQ ID NO: 54 or a variant thereof having at least 80% sequence identity.

[0577] In another aspect, the use of an sdAb according to any of the above embodiments to couple a target entity to an ALFA-tag is provided, wherein the ALFA-tag is suitably incorporated into the particles, preferably lipid nanoparticles.

[0578] "Heavy chain variable region" or "VH" refers to a segment of the heavy chain of an antigen-binding domain or antibody containing three CDRs inserted between flanking segments called frame regions. The frame regions are more conserved than the CDRs and form the backbone supporting them. "Light chain variable region" or "VL" refers to a segment of the light chain of an antigen-binding domain or antibody containing three CDRs inserted between frame regions.

[0579] The "complementarity-determining region" or "CDR" of an antigen-binding domain or antibody or its antigen-binding fragment refers to a highly variable loop in the variable region of the heavy or light chain of the antibody. The CDR can interact with the antigen conformation and largely determines binding to the antigen (although some framework regions are known to be involved in binding). Both the heavy chain and light chain variable regions contain three CDRs (heavy chain CDRs 1, 2, and 3, and light chain CDRs 1, 2, and 3, numbered from the amino to the carboxyl terminus).

[0580] Many definitions of CDRs are commonly used. The Kabat definition, based on sequence variability, is the most commonly used (see http: / / www.bioinf.org.uk / abs / ). The ImMunoGeneTics Information System (IMGT) (see http: / / www.imgt.org) can also be used. According to this system, the complementarity-determining region (CDR-IMGT), defined by the unique IMGT number of the V domain, is a ring region of a variable domain. There are three CDR-IMGTs in a variable domain: CDR1-IMGT (ring BC), CDR2-IMGT (ring C′C″), and CDR3-IMGT (ring FG). Other definitions of CDRs have also been developed, such as the Chothia, AbM, and contact definitions (see http: / / www.imgt.org). The CDR of the sdAb according to the invention can be defined using any suitable system, such as any suitable system known in the art.

[0581] The CDR according to the invention, defined using alternative systems known in the art, is shown in [the figure]. Figure 8 –In Table 1.

[0582] The “Chothia-Nanobody” system is based on the Chothia numbering scheme, modified to interpret the structure of nanobodies / VHHs. In the Chothia-Nanobody system, the VHH sequences are numbered according to the Chothia numbering scheme, where CDR1 is defined as H26-H35, CDR2 as H51-H60, and CDR3 as H93-102.

[0583] The above describes the IMGT and Kabat annotation systems.

[0584] "Humanized antibody" can refer to a non-human antibody that has been genetically engineered to contain a human antibody constant domain and a non-human variable domain, wherein the non-human variable domain is modified to contain a high level of sequence homology with the human variable domain.

[0585] "Humanized antibody" can also refer to an sdAb that has been modified to contain a high level of sequence homology with human variable domains. This can be achieved by grafting three (or six) non-human antibody complementarity-determining regions (CDRs) that together form the antigen-binding site onto a homologous human receptor frame region (FR). Non-limiting examples of antibody humanization methods include CDR grafting and surface remodeling (i.e., replacing surface residues to obtain a "more human" surface) and germline-based humanization. For example, to completely reconstruct the binding affinity and specificity of a parent antibody, it may be necessary to replace frame residues from the parent antibody (i.e., the non-human antibody) into a human frame region (reversion mutation). Structural homology modeling can help identify amino acid residues in the frame region that are important for the antibody's binding properties. Therefore, a humanized antibody may contain a non-human CDR sequence, primarily a human frame region optionally containing one or more amino acid reversion mutations of a non-human amino acid sequence, and optionally, a fully human constant region. Optionally, additional amino acid modifications, not necessarily reversion mutations, may be introduced to obtain a humanized antibody with preferred characteristics such as affinity and biochemical properties. Humanization of nonhuman therapeutic antibodies is performed to minimize their immunogenicity in humans, while such humanized antibodies maintain the specificity and binding affinity of the nonhuman-derived antibodies. Exemplary methods for humanization of VHH are described in Vincke et al. (Journal of Biological Chemistry; 2009; 284(5); 3273-3284) and Rossotti et al. (FEBS; 2021; doi:10.1111 / febs / 15809).

[0586] Appropriately, the main target portion may include Table 1 (see Table 1). Figure 8 Combinations of CDR1, CDR2, and CDR3 listed in Table 1 are included. Specifically, the primary target portion may contain CDRs comprising the following: Figure 8 The sequences of CDR1, CDR2, and CDR3 listed in Table 1. Suitablely, at least one of the CDRs listed in Table 1 may contain one, two, or three amino acid mutations.

[0587] Suitable, sdAb may include Table 1 (see Table 1). Figure 8 Combinations of CDR1, CDR2, and CDR3 listed in Table 1. Specifically, sdAb may contain CDRs that include those listed in Table 1. Figure 8 The sequences of CDR1, CDR2, and CDR3 listed in Table 1. Suitablely, at least one of the CDRs listed in Table 1 may contain one, two, or three amino acid mutations.

[0588] Appropriately, combinations of CDR1, CDR2, and CDR3 can be selected from those shown in Table 1 ( Figure 8 The CDRs listed in Table 1 are CDR1, CDR2, and CDR3; each of the CDRs is defined using the same annotation system. Suitablely, each of the CDRs may be a CDR as defined by the "Chothia-Nanobody" annotation system. Suitablely, each of the CDRs may be a CDR as defined by the IMGT annotation system. Suitablely, each of the CDRs may be a CDR as defined by the Kabat annotation system. Suitablely, at least one of the CDRs listed in Table 1 may contain one, two, or three amino acid mutations.

[0589] Suitablely, a CDR may comprise the CDR sequences shown in Table 1. Suitablely, a CDR may consist of the CDR sequences shown in Table 1.

[0590] sdAb may include CDR1 selected from the group consisting of SEQ ID NO: 1, 4, 9, 12, 15, 18, 21, 23, 25, 29 and 32; CDR2 selected from the group consisting of SEQ ID NO: 2, 5, 7, 10, 13, 16, 19, 22, 24, 26, 30 and 33; and CDR3 selected from the group consisting of SEQ ID NO: 3, 6, 8, 11, 14, 17, 20, 27, 28, 31 and 34.

[0591] Suitable, the primary target portion or sdAb may contain a CDR with the following sequence:

[0592] CDR1 – GFTFEDYAIG (SEQ ID NO: 1)

[0593] CDR2 – IRNRDGSTYYK (SEQ ID NO: 2)

[0594] CDR3 – AALKLGRLLGLVHMPAQYEYDY (SEQ ID NO: 3);

[0595] Optionally, one or more of the CDRs may contain one, two, or three amino acid mutations.

[0596] Suitable, the primary target portion or sdAb may contain a CDR with the following sequence:

[0597] CDR1 – GFTFDDYAIG (SEQ ID NO: 4)

[0598] CDR2 – IQNKDGSTYYK (SEQ ID NO: 5)

[0599] CDR3 – AALKLGRLLRLVHMPAQYEYDY (SEQ ID NO: 6);

[0600] Optionally, one or more of the CDRs may contain one, two, or three amino acid mutations.

[0601] Suitable, the primary target portion or sdAb may contain a CDR with the following sequence:

[0602] CDR1 – GFTFDDYAIG (SEQ ID NO: 4)

[0603] CDR2 – ISIRNGKTYYS (SEQ ID NO: 7)

[0604] CDR3 – AALKLGLGLVYLPAQYEYDY (SEQ ID NO: 8);

[0605] Optionally, one or more of the CDRs may contain one, two, or three amino acid mutations.

[0606] sdAb may include CDR1 selected from SEQ ID NO: 1 and 4; CDR2 selected from the group consisting of SEQ ID NO: 2, 5 and 7; and CDR3 selected from the group consisting of SEQ ID NO: 3, 6 and 8.

[0607] The main target portion (preferably VHH) or sdAb of a CDR containing one, two or three amino acid mutations compared to one or more reference CDRs in Table 1 maintains the ability to bind IgD.

[0608] As will be apparent, any CDR defined in this document by the “Chothia-Nanobody” system can be replaced by the corresponding CDR defined by the IMGT or Kabat system.

[0609] Suitablely, the primary target region is VHH, which contains, as in SEQ ID NO: 35-48 (see...). Figure 9 –A sequence shown in any of the items in Table 2) or a variant thereof that has at least 80% sequence identity with it.

[0610] Suitablely, the present invention provides a VHH comprising, as in SEQ ID NO: 35-48 (see SEQ ID NO: 35-48). Figure 9 –A sequence shown in any of the items in Table 2) or a variant thereof that has at least 80% sequence identity with it.

[0611] Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 35 or a variant having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. For example, VHH may comprise the sequence shown in SEQ ID NO: 35 or a variant having at least 90% sequence identity with it. Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 35.

[0612] Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 36 or a variant having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. For example, VHH may comprise the sequence shown in SEQ ID NO: 36 or a variant having at least 90% sequence identity with it. Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 36.

[0613] Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 37 or a variant having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. For example, VHH may comprise the sequence shown in SEQ ID NO: 37 or a variant having at least 90% sequence identity with it. Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 37.

[0614] Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 38 or a variant having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. For example, VHH may comprise the sequence shown in SEQ ID NO: 38 or a variant having at least 90% sequence identity with it. Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 38.

[0615] Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 39 or a variant having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. For example, VHH may comprise the sequence shown in SEQ ID NO: 39 or a variant having at least 90% sequence identity with it. Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 39.

[0616] Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 40 or a variant having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. For example, VHH may comprise the sequence shown in SEQ ID NO: 40 or a variant having at least 90% sequence identity with it. Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 40.

[0617] Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 41 or a variant having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. For example, VHH may comprise the sequence shown in SEQ ID NO: 41 or a variant having at least 90% sequence identity with it. Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 41.

[0618] Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 42 or a variant having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. For example, VHH may comprise the sequence shown in SEQ ID NO: 42 or a variant having at least 90% sequence identity with it. Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 42.

[0619] Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 43 or a variant having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. For example, VHH may comprise the sequence shown in SEQ ID NO: 43 or a variant having at least 90% sequence identity with it. Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 43.

[0620] Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 44 or a variant having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. For example, VHH may comprise the sequence shown in SEQ ID NO: 44 or a variant having at least 90% sequence identity with it. Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 44.

[0621] Suitablely, VHH may comprise the VHH sequence shown in SEQ ID NO: 45 or a variant having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. For example, VHH may comprise the sequence shown in SEQ ID NO: 45 or a variant having at least 90% sequence identity with it. Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 45.

[0622] Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 46 or a variant having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. For example, VHH may comprise the sequence shown in SEQ ID NO: 46 or a variant having at least 90% sequence identity with it. Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 46.

[0623] Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 47 or a variant having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. For example, VHH may comprise the sequence shown in SEQ ID NO: 47 or a variant having at least 90% sequence identity with it. Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 47.

[0624] Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 48 or a variant having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. For example, VHH may comprise the sequence shown in SEQ ID NO: 48 or a variant having at least 90% sequence identity with it. Suitablely, VHH may comprise the sequence shown in SEQ ID NO: 48.

[0625] The term "variant" refers to a polypeptide that has the same function as the amino acid sequence described herein but includes one or more amino acid substitutions, insertions, or deletions. Therefore, a VHH may contain variants that have at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the sequence described herein.

[0626] As used herein, “variant” is synonymous with “mutant” and refers to an amino acid sequence that differs from the corresponding wild-type sequence. The term “wild-type” is used to mean a protein that has the same amino acid sequence as the natural protein.

[0627] One or more mutations (substitution, addition, or deletion) can be introduced into each CDR without negatively affecting binding activity. For example, each CDR can have one, two, or three amino acid mutations. A VHH including an IgD-binding domain containing one or more of these CDRs (which contain one, two, or three amino acid mutations) can appropriately maintain the ability to bind IgD.

[0628] The ability of IgD binding domains to bind IgD can be assessed by determining binding affinity. Quantitative assessment or measurement of binding affinity (e.g., establishing KD values) can be determined or measured using methods known in the art, such as by biolayer interferometry (BLI) or surface plasmon resonance, for example, using the Biacore® system. In addition to the equilibrium dissociation constant (KD), the association rate constant (Ka(1 / Ms)) and the dissociation rate constant (KD(1 / s)) can also be determined.

[0629] Surface plasmon resonance (SPR) experiments can be performed using, for example, the Biacore T200.

[0630] Methods for determining binding specificity include, but are not limited to, ELISA, Western blotting, immunohistochemistry, flow cytometry, Foster resonance energy transfer (FRET), phage display libraries, yeast double-hybrid screening, immunoprecipitation, bimolecular fluorescence complementation, and tandem affinity purification. Binding affinity can also be determined using methods such as fluorescence quenching and isothermal titration calorimetry.

[0631] Compared to the corresponding full-length sequence, the variants covered by this invention have substantially the same or improved binding activity.

[0632] Identity comparisons can be performed visually, or more generally, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percentage of identity between two or more sequences. A suitable computer program for performing such comparisons is the GCG Wisconsin Bestfit software package (University of Wisconsin, USA; Devereux et al., 1984, Nucleotide sequences Research 12:387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST software package (see Ausubel et al., 1999 ibid. – Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410), and the GNEWORKS comparison tool suite. Both BLAST and FASTA can be used for offline and online retrieval. For example, the percentage of identity between two polypeptide sequences can be easily determined using BLAST (which is available free of charge at http: / / blast.ncbi.nlm.nih.gov).

[0633] Once the software has generated the best alignment, it can calculate the identity percentage. The software typically uses this as part of the sequence comparison and generates numerical results.

[0634] Sequences may have one or more deletions, insertions, or substitutions of amino acid residues that produce silencing changes and result in functionally equivalent molecules. These sequences are encompassed within this invention. Intentional amino acid substitutions can be made based on the similarity of residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties, provided that activity is preserved. For example, 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.

[0635] Advantageously, the sdAb of the present invention is capable of specifically binding to both human and rhesus monkey (Macaca mulatta) IgD. This property can be beneficial, for example, in terms of availability in preclinical animal models.

[0636] Compared to other immunoglobulins, the sdAb of the present invention may have preferential or selective binding to IgD. Suitably, the sdAb can bind IgD but does not bind or is substantially unable to bind one or more other immunoglobulins. For example, the sdAb of the present invention may not or may be substantially unable to bind IgA, IgE, IgG, and / or IgM. The sdAb may not or may be substantially unable to bind IgA, IgE, IgG, and IgM. IgA, IgE, IgG, and / or IgM may be human or rhesus monkey immunoglobulins.

[0637] The ability to bind IgD and / or the inability or substantially inability to bind immunoglobulins other than IgD can be determined using methods known in the art. Methods for determining binding specificity include, but are not limited to, ELISA, Western blotting, immunohistochemistry, flow cytometry, Foster resonance energy transfer (FRET), phage display library, yeast double heterozygous screening, immunoprecipitation, bimolecular fluorescence complementation, and tandem affinity purification.

[0638] Suitably, the sdAb of the present invention can be internalized into the cell after binding to IgD on the cell surface. For example, this can be advantageous for the delivery of the load to the target cell. The internalization of the sdAb can be determined using methods known in the art, such as confocal microscopy and flow cytometry. Suitably, the cell can be a B cell.

[0639] Suitablely, the sdAb of the present invention can activate B cells after binding to IgD on the surface of B cells. B cell activation can be determined using methods known in the art; for example, FACS analysis of surface B cell activation markers such as CD86 is known.

[0640] Suitably, the sdAb of the present invention can retain its ability to bind IgD, particularly IgD expressed on the cell surface, in the presence of serum (e.g., human serum). The ability to bind IgD in the presence of serum can be determined using methods known in the art (e.g., ELISA). For example, binding to IgD can be detectable even in the presence of serum. Retaining the ability to bind IgD in the presence of serum can mean that, compared to the level of IgD binding in the absence of serum, the level of IgD binding 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%. For example, retaining the ability to bind IgD in the presence of serum can mean that the level of IgD binding is reduced by less than 80%, optionally less than 50%, in the presence of serum. The ability to retain IgD binding in the presence of serum can mean that the level of IgD binding 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%, or at least 80% of the level of IgD binding in the absence of serum. For example, the ability to retain IgD binding in the presence of serum can mean that the level of IgD binding is at least 10%, optionally at least 20%, of the level of IgD binding detected in the absence of serum. Suitably, the ability to retain IgD binding in the presence of serum can refer to the level of IgD binding in the presence of 50% human serum; suitably based on incubation at 37°C for 30 minutes (as described in the embodiments of the invention). Suitably, sdAb can be provided on the surface of lipid nanoparticles (e.g., as described in the embodiments of the invention).

[0641] Humanized ALFA-label VHH

[0642] The present invention also provides a humanized sdAb, preferably VHH, that can bind to ALFA-tagged peptides.

[0643] Suitable, the humanized VHH capable of binding to the ALFA-tagged peptide may contain a CDR comprising the following sequence:

[0644] CDR1 – GVTISALNAMAMG (SEQ ID NO: 49)

[0645] CDR2 – AVSERGNTY (SEQ ID NO: 53)

[0646] CDR3 – LEDRVDSFHDY (SEQ ID NO: 51)

[0647] (According to the AbM definition) Optionally, one or more of the CDRs contain one, two or three amino acid mutations.

[0648] CDRs for humanized VHHs that can bind to ALFA-tagged peptides can also be provided based on IMGT or Kabat annotations.

[0649] Appropriately, according to the Kabat annotation, the second associative domain may contain a CDR containing the following sequence:

[0650] CDR1 – ALNAMAMG (SEQ ID NO: 137)

[0651] CDR2 – AVSERGNTYYRDSVKG (SEQ ID NO: 138)

[0652] CDR3 – LEDRVDSFHDY (SEQ ID NO: 139);

[0653] Optionally, one or more of the CDRs may contain one, two, or three amino acid mutations.

[0654] Appropriately, according to the IMGT annotation, the second associative domain may contain a CDR containing the following sequence:

[0655] CDR1 – GVTISALNAMA (SEQ ID NO: 141)

[0656] CDR2 – VSERGNT (SEQ ID NO: 53)

[0657] CDR3 – HVLEDRVDSFHDY (SEQ ID NO: 142);

[0658] Optionally, one or more of the CDRs may contain one, two, or three amino acid mutations.

[0659] It should be understood that the second binding domain of one, two, or all three of the mutations introduced into the CDR sequence can still bind specifically to the ALFA tag.

[0660] Suitable, the ALFA tag can be an ALFA-tagged peptide as described herein.

[0661] VHH may contain SEQ ID NO: 54 or a variant thereof having at least 80% sequence identity. For example, VHH may contain the sequence shown in SEQ ID NO: 54 or a variant thereof having at least 90% sequence identity. Suitably, VHH may contain the sequence shown in SEQ ID NO: 54.

[0662] Appropriately, the variants maintain the ability to bind to the ALFA tag. Suitable variants are described in this article.

[0663] The present invention also provides the use of humanized sdAb as described above, preferably VHH as described above, to couple a target entity to an ALFA-tag, suitably wherein the ALFA-tag is incorporated into particles, preferably lipid nanoparticles.

[0664] Therefore, the humanized sdAb of the present invention is applicable for use in docking compounds as described herein. It will be apparent, however, that the humanized sdAb of the present invention is not limited to use in docking compounds containing a portion capable of binding IgD. For example, the humanized sdAb of the present invention can be used in docking compounds containing a targeting portion capable of binding any target (e.g., any target cell surface target).

[0665] docking compounds

[0666] In some aspects, the functionalized particles of the present invention contain a docking compound. In one embodiment, the docking compound is a compound of formula (I) as defined herein.

[0667] In some embodiments, a docking compound (e.g., a compound of formula (I)) is used to form a linker compound (i.e., a linker compound of formula (A') that is integrated into a particle containing a nucleic acid payload for delivery to the target cell. In some embodiments, the linker compound is a non-covalent linker to the docking compound. In some embodiments, the linker compound and the linker compound are either non-covalent or covalent. In some embodiments, the linker compound includes a binding portion for covalently attaching to a hydrophobic portion (e.g., a lipid) for binding to the docking compound. The hydrophobic portion (e.g., a lipid) forms part of the particle.

[0668] In some embodiments, the docking compound includes a “primary targeting portion” as defined above (e.g., B3 of a compound of formula (I)), such as a portion that targets IgD on target cells. In some embodiments, as used herein, a “primary targeting portion” refers to a portion of the docking compound capable of binding to or binding to IgD.

[0669] The docking compound also includes a group (as defined herein, portion B2) that acts as a binding partner for a corresponding binding portion B1 of a linker compound of formula (A) as defined herein. A portion of the linker compound containing a hydrophobic portion (e.g., lipid) (having a covalently attached binding portion of the docking compound) is integrated into the load-carrying particle, thereby forming a connection between the particle and the docking compound. The portion of the docking compound bound to the linker compound and the main targeting portion are preferably connected to each other by covalent bonds.

[0670] According to some embodiments, the docking compound comprises a bispecific molecule, such as a bispecific polypeptide, for example, a bispecific antibody. In some embodiments, the docking compound comprises a binding domain capable of binding to IgD and a binding domain capable of binding to a linker compound. In some embodiments, 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 linker compound. In some embodiments, at least one binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of the antibody. In some embodiments, each binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of the antibody. In some embodiments, at least one binding domain comprises a single-domain antibody, such as VHH. In some embodiments, each binding domain comprises a single-domain antibody, such as VHH. In some embodiments, one binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of the antibody, and other binding domains comprise single-domain antibodies, such as VHH. In some embodiments, the binding domain that binds to the primary target comprises the heavy chain variable region (VH) and light chain variable region (VL) of the antibody. In some embodiments, the binding domain capable of binding to IgD comprises a single-domain antibody, such as VHH. In some embodiments, the binding domain capable of binding to a linker compound comprises the heavy chain variable region (VH) and light chain variable region (VL) of the antibody. In some embodiments, the binding domain capable of binding to a linker compound comprises a single-domain antibody, such as VHH.

[0671] In some embodiments, the docking compound comprises a fusion protein having a binding domain capable of binding to IgD and a binding domain capable of binding to the linker compound.

[0672] In some embodiments, the docking compound comprises a single peptide chain. In some embodiments, the single peptide chain comprises a portion capable of binding to IgD (e.g., an antibody, antibody fragment, or DARPin) and a portion capable of binding to the linker compound (e.g., an antibody or antibody fragment). In some embodiments, the antibody fragment is VHH, scFv, or a mixture thereof. In various embodiments, the docking compound comprises one of the following structures (from the N-terminus to the C-terminus):

[0673] VHH (α-linker compound) - optional linker - VHH (α-IgD)

[0674] VHH (α IgD) - optional linker - VHH (α linker compound)

[0675] VHH (α-linker compound) - optional linker - scFv (α IgD)

[0676] scFv (α IgD) - optional linker - VHH (α linker compound)

[0677] VHH (α IgD) - optional linker - scFv (α linker compound)

[0678] scFv (α-linker compound) - optional linker - VHH (α IgD)

[0679] scFv (α-linker compound) - optional linker - scFv (α IgD)

[0680] scFv (α IgD) - optional linker - scFv (α linker compound)

[0681] In some embodiments, the docking compound comprises a bispecific molecule, such as a bispecific peptide, for example, a bispecific antibody, one specificity of which binds to an epitope tag (e.g., an ALFA-tag), and the other specificity of which binds to IgD, for example, on a target cell. In some embodiments, the specificity of binding to the epitope tag is an antibody or antibody fragment, such as an NbALFA-nanobody (NbALFA). In some embodiments, the specificity of binding to IgD is an antibody, antibody fragment, or DARPin. In some embodiments, the IgD-targeting portion is selected from the group consisting of anti-IgD DARPin, anti-IgDVHH, and anti-IgDscFv, and / or the portion of the docking compound that binds to the linker compound is an NbALFA-nanobody (NbALFA). In some embodiments, the docking compound has a structure selected from the group consisting of NbALFA x anti-IgDDARPin, NbALFA x anti-IgDVHH, and NbALFA x anti-IgDscFv. In some embodiments, the docking compound comprises a nanobody that binds to an epitope tag (e.g., an ALFA-tag) and a bispecific antibody against -IgD VHH. In some embodiments, the docking compound comprises a nanobody that binds to an epitope tag (e.g., an ALFA-tag) and a bispecific antibody against -IgD scFv. In some embodiments, the docking compound comprises a bispecific molecule that binds to an epitope tag (e.g., an ALFA-tag) and anti-IgD DARPin.

[0682] Suitable, the docking compound may contain a linker group that connects components B2 and B3.

[0683] Suitable linkers are well known in the art, for example, serine-glycine linkers.

[0684] Suitablely, the serine-glycine linker may contain or consist of SGGGGS (SEQ ID NO: 76) or GGGGSGGGS (SEQ ID NO: 77).

[0685] Suitably, the docking compound may comprise, or consist of, any of the sequences in SEQ ID NO: 58-74 or variants having at least 80% identity with them (SEQ ID NO: 58-74 shown in...). Figure 10 -In Table 3).

[0686] Suitably, the docking compound may comprise, or consist of, the sequence of SEQ ID NO: 58 or a variant thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. Suitably, the docking compound may comprise, or consist of, the sequence shown in SEQ ID NO: 58.

[0687] Suitably, the docking compound may comprise, or consist of, the sequence of SEQ ID NO: 59 or a variant thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. Suitably, the docking compound may comprise, or consist of, the sequence shown in SEQ ID NO: 59.

[0688] Suitably, the docking compound may comprise, or consist of, the sequence of SEQ ID NO: 60 or a variant thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. Suitably, the docking compound may comprise, or consist of, the sequence shown in SEQ ID NO: 60.

[0689] Suitably, the docking compound may comprise, or consist of, the sequence of SEQ ID NO: 61 or a variant thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. Suitably, the docking compound may comprise, or consist of, the sequence shown in SEQ ID NO: 61.

[0690] Suitably, the docking compound may comprise, or consist of, the sequence of SEQ ID NO: 62 or a variant thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. Suitably, the docking compound may comprise, or consist of, the sequence shown in SEQ ID NO: 62.

[0691] Suitably, the docking compound may comprise, or consist of, the sequence of SEQ ID NO: 63 or a variant thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. Suitably, the docking compound may comprise, or consist of, the sequence shown in SEQ ID NO: 63.

[0692] Suitably, the docking compound may comprise, or consist of, the sequence of SEQ ID NO: 64 or a variant thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. Suitably, the docking compound may comprise, or consist of, the sequence shown in SEQ ID NO: 64.

[0693] Suitably, the docking compound may comprise, or consist of, the sequence of SEQ ID NO: 65 or a variant thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. Suitably, the docking compound may comprise, or consist of, the sequence shown in SEQ ID NO: 65.

[0694] Suitably, the docking compound may comprise, or consist of, the sequence of SEQ ID NO: 66 or a variant thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. Suitably, the docking compound may comprise, or consist of, the sequence shown in SEQ ID NO: 66.

[0695] Suitably, the docking compound may comprise, or consist of, the sequence of SEQ ID NO: 67 or a variant thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. Suitably, the docking compound may comprise, or consist of, the sequence shown in SEQ ID NO: 67.

[0696] Suitably, the docking compound may comprise, or consist of, the sequence of SEQ ID NO: 68 or a variant thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. Suitably, the docking compound may comprise, or consist of, the sequence shown in SEQ ID NO: 68.

[0697] Suitably, the docking compound may comprise, or consist of, the sequence of SEQ ID NO: 69 or a variant thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. Suitably, the docking compound may comprise, or consist of, the sequence shown in SEQ ID NO: 69.

[0698] Suitably, the docking compound may comprise, or consist of, the sequence of SEQ ID NO: 70 or a variant thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. Suitably, the docking compound may comprise, or consist of, the sequence shown in SEQ ID NO: 70.

[0699] Suitably, the docking compound may comprise, or consist of, the sequence of SEQ ID NO: 71 or a variant thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. Suitably, the docking compound may comprise, or consist of, the sequence shown in SEQ ID NO: 71.

[0700] Suitably, the docking compound may comprise, or consist of, the sequence of SEQ ID NO: 72 or a variant thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. Suitably, the docking compound may comprise, or consist of, the sequence shown in SEQ ID NO: 72.

[0701] Suitably, the docking compound may comprise, or consist of, the sequence of SEQ ID NO: 73 or a variant thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. Suitably, the docking compound may comprise, or consist of, the sequence shown in SEQ ID NO: 73.

[0702] Suitably, the docking compound may comprise, or consist of, the sequence of SEQ ID NO: 74 or a variant thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with it. Suitably, the docking compound may comprise, or consist of, the sequence shown in SEQ ID NO: 74.

[0703] Interaction regions on linker compounds and docking compounds

[0704] In some embodiments, portions of the linker compound that interact with each other (e.g., portion B1 of the compound of formula (A), covalently attached to the hydrophobic portion or the binding portion of the anionic polymer) and portions of the docking compound (e.g., portion B2 of the compound of formula (I), bound to the binding portion covalently attached to the hydrophobic portion or the anionic polymer) are, for example, non-covalently bonded to each other.

[0705] In some embodiments, portions of the linker compound and the docking compound that interact with each other (e.g., portion B1, formula (A) and portion B2, formula (I)) bind to each other under physiological conditions.

[0706] In some embodiments, the portions of the linker compound and the docking compound that interact with each other (e.g., portion B1, formula (A) and portion B2, formula (I)) are antibody / antigen systems.

[0707] In some embodiments, a portion of the linker compound that binds to the docking compound (e.g., portion B1, formula (A)) contains a peptide or protein, such as a peptide tag, and a portion of the docking compound that binds to the linker compound (e.g., portion B2, formula (I)) contains a binder that binds to the peptide or protein, such as an antibody or antibody fragment.

[0708] In some embodiments, a portion of the docking compound that binds to the linker compound (e.g., portion B2, formula (I)) contains a peptide or protein, such as a peptide tag, and a portion of the linker compound that binds to the docking compound (e.g., portion B1, formula (A)) contains a binder that binds to the peptide or protein, such as an antibody or antibody fragment.

[0709] In some implementations, portion B1 contains an antibody, an antibody-like molecule, or a VHH, and portion B2 is a peptide.

[0710] In some implementations, portion B2 contains an antibody, an antibody-like molecule, or a VHH, and portion B1 is a peptide.

[0711] In some embodiments, portion B1 of the compound of formula (A) contains a peptide tag, and portion B2 of the compound of formula (I) binds to the compound of formula (A) via the peptide tag.

[0712] In some implementations, the peptide tag includes an ALFA-tag.

[0713] In some embodiments, part B2 of the compound of formula (I) is a VHH that can bind to the ALFA-tag.

[0714] In some embodiments, portion B2 of the compound of formula (I) contains a peptide tag, and portion B1 of the compound of formula (A) binds to the compound of formula (I) via the peptide tag.

[0715] In some implementations, the peptide tag includes an ALFA-tag.

[0716] In some embodiments, part B1 of the compound of formula (A) is a VHH that can bind to the ALFA-tag.

[0717] In some embodiments, portions of the linker compound and docking compound that interact with each other (e.g., portion B1, formula (A) and portion B2, formula (I)) contain an epitope tag / binding agent system.

[0718] As used in this article, an "epitaph tag" refers to an amino acid segment that an antibody or a protein molecule with antibody-like function can bind to.

[0719] In some embodiments, the epitope tag includes an ALFA-tag. In some embodiments, the epitope tag / binding agent system includes an ALFA-tag and an ALFA-specific single-domain antibody (sdAb) or an NbALFA-nanobody. The ALFA-tag can be defined as described below.

[0720] polymer

[0721] In some aspects of this disclosure, the functionalized particles of the present invention comprise polymers. In this specification, the term "polymer" is given its conventional meaning, for example, a molecular structure comprising one or more repeating units (monomers) linked by covalent bonds. The repeating units may be all identical, or in some cases, more than one type of repeating unit may be present within the polymer. In some cases, the polymer is of biological origin, i.e., a biopolymer, such as a protein. In some cases, additional portions, such as targeting portions, may also be present in the polymer.

[0722] Polymers are commonly used materials for nanoparticle-based delivery due to their high chemical flexibility. Typically, cationic polymers are used to electrostatically aggregate negatively charged nucleic acids into nanoparticles. These positively charged groups are often composed of amines, which alter their protonation state in a pH range of 5.5 to 7.5, believed to induce ionic imbalance, leading to endosome disruption.

[0723] Polymers such as poly-L-lysine, polyamide amine, protamine, and polyethyleneimine, as well as naturally occurring polymers such as chitosan, have all been used in nucleic acid delivery and are suitable as cationic polymers for this paper. Furthermore, some researchers have synthesized polymers specifically for nucleic acid delivery. In particular, poly(β-amino esters) are widely used in nucleic acid delivery due to their ease of synthesis and biodegradability. Such synthetic polymers are also suitable as cationic polymers for this paper.

[0724] A polymer is called a "polymer" if it contains more than one type of repeating unit. It should be understood that the polymer used herein can be a copolymer. The repeating units forming a copolymer can be arranged in any manner. For example, the repeating units can be arranged in a random order, an alternating order, or as a "block" copolymer (i.e., comprising one or more regions each containing a first repeating unit (e.g., a first block) and one or more regions each containing a second repeating unit (e.g., a second block)). Block copolymers can have two (diblock copolymers), three (triblock copolymers), or more different blocks.

[0725] In some embodiments, the polymer is biocompatible. A biocompatible polymer is one that typically does not cause significant cell death at moderate concentrations. In some embodiments, the biocompatible polymer is biodegradable, i.e., the polymer is capable of chemically and / or biologically degrading within physiological environments, such as in vivo.

[0726] In some embodiments, the polymer may be protamine or polyolefin. In some embodiments, the particulate-forming component may comprise protamine or polyolefin. The term "protamine" refers to a variety of strongly basic proteins of relatively low molecular weight, rich in arginine, and particularly found in the sperm cells of various animals (such as fish) that substitute for histones in DNA association. Specifically, the term "protamine" refers to proteins found in fish sperm that are strongly basic, soluble in water, coagulate without heating, and yield primarily arginine upon hydrolysis. In purified forms, they are used in long-acting formulations of insulin and to neutralize the anticoagulant effect of heparin.

[0727] According to this disclosure, as used herein, the term "protamine" is intended to include 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 fragments thereof, as well as artificial (synthetic) polypeptides that are specifically designed for a particular purpose and cannot be isolated from natural or biological sources.

[0728] In some embodiments, the polyolefin imide comprises polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine. A preferred polyolefin imide is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75. 10 2 Up to 10 7 Da, preferred 1000 to 10 5 Da, more preferably 10,000 to 40,000 Da, more preferably 15,000 to 30,000 Da, and even more preferably 20,000 to 25,000 Da.

[0729] According to this disclosure, linear polyolefin imides, such as linear polyethyleneimine (PEI), are preferred.

[0730] Stealth Polymer

[0731] In some embodiments, the polymer is a hydrophilic polymer and the linker compound comprises an amphiphilic derivative of the polymer. In some embodiments, the amphiphilic derivative of the polymer comprises a hydrophobic component (e.g., a lipid component) that allows it to anchor within the particles and a hydrophilic component of the polymer that imparts hydrophilic properties to the exterior of the particles at its surface. In some embodiments, the amphiphilic derivative of the polymer is inserted into the particles via its hydrophobic end.

[0732] Therefore, the polymer component faces the exterior of the particle and forms a protective hydrophilic shell surrounding the particle. In some embodiments, the polymer portion of the amphiphilic derivative contributes to cloaking properties on the particle. In some embodiments, the polymer portion of the amphiphilic derivative imparts cloaking properties to the particle. In some embodiments, the plasma half-life of the particles described herein is greater than 2 hours, for example, 3 to 10 hours. This property advantageously allows the particles to accumulate at target cells and release their contents (load) within a reasonable timeframe. Therefore, the effectiveness of the targeted delivery described herein is improved.

[0733] The term “stealth” is used herein to describe the ability of particles described herein to be undetectable by the immune system of the host to which they are applied and then isolated and / or degraded, or to be barely detected and then isolated and / or degraded, and / or to be detected and then later isolated and / or degraded.

[0734] In some embodiments, 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.

[0735] In some embodiments, the polymer is designed to spatially stabilize the particles by forming a protective hydrophilic layer. In some embodiments, when such particles are administered in vivo, the polymer can reduce particle association with serum proteins and / or uptake by the reticuloendothelial system.

[0736] In some embodiments, the polymer is PEG, and the PEG is a linear or branched polymer of ethylene glycol or ethylene oxide, optionally substituted. In some embodiments, the PEG is unsubstituted. In some embodiments, the PEG is substituted, for example, with one or more alkyl, alkoxy, acyl, hydroxyl, or aryl groups. In some embodiments, the PEG has a molecular weight of 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 6,000, in another embodiment about 150 to about 5,000, in another embodiment about 150 to about 4,000, in another embodiment about 150 to about 3,000, in another embodiment about 300 to about 3,000, in another embodiment about 1,000 to about 3,000, and in yet another embodiment about 1,500 to about 2,500.

[0737] In some embodiments, the PEG moiety of the amphiphilic derivative of the polymer has a molecular weight of 1000 or greater. In some embodiments, the PEG moiety of the amphiphilic derivative of the polymer comprises the formula (O-CH2-CH2). n (where n is the number of ethylene oxide units) 2 or more units, such as 5 or more units, such as 10 or more units. In some embodiments, the PEG contains 20 to 200 ethylene oxide units, such as about 45 ethylene oxide units.

[0738] In some implementations, PEG contains “PEG2k”, also known as “PEG 2000”, which has an average molecular weight of about 2,000 Daltons.

[0739] In some implementations, DSPE-PEG2000, DSPE-PEG3000 and DSPE-PEG5000 are used as amphiphilic derivatives of the polymer.

[0740] In some embodiments, the polymer is pSar and pSar contains 2 to 200 sarcosine units, such as 5 to 100 sarcosine units, 10 to 50 sarcosine units, 15 to 40 sarcosine units, for example, about 23 sarcosine units.

[0741] In some implementations, pSar includes the structure of the following general formula:

[0742]

[0743] Where s is the number of sarcosine units.

[0744] In some embodiments, the polymer is POX and / or POZ, and the POX and / or POZ polymer comprises 2 to 200, 2 to 190, 2 to 180, 2 to 170, 2 to 160, 2 to 150, 2 to 140, 2 to 130, 2 to 120, 2 to 110, 2 to 100, 2 to 90, 2 to 80, 2 to 70, 5 to 200, 5 to 190, 5 to 180, 5 to 170, 5 to 160, 5 to 150. 5 to 140, 5 to 130, 5 to 120, 5 to 110, 5 to 100, 5 to 90, 5 to 80, 5 to 70, 10 to 200, 10 to 190, 10 to 180, 10 to 170, 10 to 160, 10 to 150, 10 to 140, 10 to 130, 10 to 120, 10 to 110, 10 to 100, 10 to 90, 10 to 80, or 10 to 70 POX and / or POZ repeating units.

[0745] In some implementations, the POX and / or POZ polymers comprise the following general formula:

[0746] ,

[0747] Where a is an integer from 1 to 2; R 11 It is an alkyl group, especially C 1-3 Alkyl groups, such as methyl, ethyl, isopropyl, or n-propyl, are chosen independently for each repeating unit; and m refers to the number of POX and / or POZ repeating units.

[0748] In some embodiments, the POX and / or POZ polymers are polymers of POX and contain repeating units of the following general formula:

[0749]

[0750] Where R 11 As defined above.

[0751] In some embodiments, the POX and / or POZ polymers are polymers of POZ and contain repeating units of the following general formula:

[0752]

[0753] Where R 11 As defined above.

[0754] In any of the above embodiments of the formula, m (i.e., the number of repeating units in the polymer) is preferably 2 to 190, such as 2 to 180, 2 to 170, 2 to 160, 2 to 150, 2 to 140, 2 to 130, 2 to 120, 2 to 110, 2 to 100, 2 to 90, 2 to 80, 2 to 70, 5 to 200, 5 to 190, 5 to 180, 5 to 170, 5 to 160, 5 to 150, 5 to 14 ... 0, 5 to 130, 5 to 120, 5 to 110, 5 to 100, 5 to 90, 5 to 80, 5 to 70, 10 to 200, 10 to 190, 10 to 180, 10 to 170, 10 to 160, 10 to 150, 10 to 140, 10 to 130, 10 to 120, 10 to 110, 10 to 100, 10 to 90, 10 to 80, or 10 to 70. In some embodiments, m is 2 to 180, such as 4 to 160, 6 to 140, 8 to 120, or 10 to 100, for example, 20 to 80, 30 to 70, or 40 to 50.

[0755] In some embodiments, the POX and / or POZ polymers are copolymers comprising repeating units of the following general formula:

[0756] ,

[0757] Where R 11 As defined above. In some embodiments, the number of repeating units shown on the left side of the copolymer is 1 to 199. In some embodiments, the number of repeating units shown on the right side of the copolymer is 1 to 199. In some embodiments, the sum of the number of repeating units shown on the left side and the number of repeating units shown on the right side of the copolymer is 2 to 200.

[0758] In some embodiments of the oxazoline- and / or oxazine-modified hydrophobic moieties (e.g., lipids), the number of repeating units of the left-hand side 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 the right-hand side 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 the left-hand side and the right-hand side in the copolymer is 2 to 180, such as 4 to 160, 6 to 140, 8 to 120, or 10 to 100, for example, 20 to 80, 30 to 70, or 40 to 50.

[0759] In some of the above embodiments, R occurs each time (i.e., in each repeating unit). 11 They can be the same alkyl group (e.g., R). 11 (The repeating unit may contain a methyl group). In some alternative embodiments, at least one repeating unit contains R. 11 Unlike R in another repeating unit 11 (For example, for at least one repeating unit, R) 11 It is a specific alkyl group (such as ethyl), and for at least one different repeating unit, R 11 These are different specific alkyl groups (such as methyl). For example, each R 11 It can be selected from two different alkyl groups (such as methyl and ethyl) and not all R 11 They are all the same alkyl group. In any of the above embodiments, R 11 Preferably, it is methyl or ethyl, more preferably methyl. Therefore, in some embodiments, each R... 11 Is it methyl or each R 11 It is ethyl. In some alternative embodiments, for each repeating unit, R 11 Independently selected from methyl and ethyl, wherein in at least one repeating unit, R 11 It is a methyl group, and in at least one repeating unit, R 11 It is an ethyl group.

[0760] In some embodiments, the polymer comprises poly-2-(2-(2-aminoethoxy)ethoxy)-acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or derivatives thereof, as defined herein.

[0761] In some embodiments, the polymer comprises the following general formula:

[0762]

[0763] in

[0764] X 11 and X 12 Together they are optionally substituted amides, optionally substituted thioamides, or esters;

[0765] Y is -CH2-, -(CH2)2-, or -(CH2)3-;

[0766] z is between 2 and 24; and

[0767] n is between 1 and 100.

[0768] In some implementation schemes,

[0769] (i) When X 11 When it is -C(O)-, then X 12 Yes -NR 1 -;

[0770] (ii) When X 11 Yes -NR 1 - when, then X 12 It is -C(O)-;

[0771] (iii) When X 11 When it is -C(S)-, then X 12 Yes -NR 1 -;

[0772] (iv) When X 11 Yes -NR 1 - when, then X 12 It is -C(S)-;

[0773] (v) When X 11 When it is -C(O)-, then X 12 It is -O-; or

[0774] (vi) When X 11 When it is -O-, then X 12 It is -C(O)-;

[0775] Where R 1 Is it hydrogen or C? 1-8alkyl.

[0776] In some implementation schemes, X 11 It is -C(O)- and X 12 Yes -NR 1 -, where R 1 Is it hydrogen or C? 1-8 Alkyl group. In some embodiments, X 11 It is -C(O)- and X 12 Yes -NR 1 -, where R 1 It is hydrogen or methyl. In some embodiments, X 11 It is -C(O)- and X 12 Yes -NR 1 -, where R 1 It is hydrogen.

[0777] In some implementations, Y is -CH2- or -(CH2)2-.

[0778] In some embodiments, the polymer comprises the following general formula:

[0779]

[0780] in

[0781] R 1 Is it hydrogen or C? 1-8 alkyl;

[0782] z is between 2 and 24; and

[0783] n is between 1 and 100.

[0784] In some embodiments of the above formula, 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.

[0785] In some embodiments, the polymer comprises the following general formula:

[0786]

[0787] in

[0788] R 1 Is it hydrogen or C? 1-8 Alkyl groups; and

[0789] n is between 1 and 100.

[0790] In some implementation schemes of the above formula, R 1 It is hydrogen or methyl. In some embodiments, R 1It is hydrogen.

[0791] In some embodiments, the polymer comprises the following general formula:

[0792]

[0793] Where n is between 1 and 100.

[0794] In some embodiments of the above formula, 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.

[0795] In some embodiments, the molar percentage of the amphiphilic derivative of the polymer integrated into the particles is 0.5 mol% to 20 mol%, preferably 1 mol% to 10 mol%, of the lipid molecules constituting the particles.

[0796] cationic polymers

[0797] In some embodiments, the functionalized particles of the present invention, particularly polymer complex (PLX) particles and esterified polymer complex (LPLX) particles, comprise a cationic polymer as a particle-forming component. In this specification, the term "cationic polymer" means a polymer as generally defined herein, carrying at least one cation, i.e., an ionic substance having a positive charge.

[0798] The cationic polymers (including polycationic polymers) contemplated for use herein include any cationic polymer capable of electrostatically binding nucleic acids. In some embodiments, the cationic polymers contemplated for use herein include any cationic polymer with which nucleic acids can associate (e.g., by forming a complex with the nucleic acid or forming vesicles in which the nucleic acid is surrounded or encapsulated).

[0799] In some embodiments, the linker compound is incorporated into the particles containing the cationic polymer by interacting the negative charge in the portion of the linker compound incorporated into the particles with the positive charge of the particles.

[0800] In some embodiments, the linker compound is incorporated into the particles containing the cationic polymer via a portion comprising the anionic polymer. In some embodiments, the cationic polymer comprises one or more of the group consisting of: cationic or polycationic peptides or proteins, including protamine, spermine or spermidine, polylysine, polyarginine; cationic polysaccharides, including chitosan; and cationic polymers, including poly(ethyleneimine), poly(propyleneimine), polyamide, polyallylamine, and polyethyleneimine. In some embodiments, the polymer comprises a polyamide amine (PAMAM) polymer.

[0801] In some embodiments, the cationic polymer is a homopolymer selected from poly(ethyleneimine), poly(propyleneimine), polyamide, polyallylamine, polyethyleneamine, polyamideamine, poly-L-lysine, poly-L-arginine, poly-L-histidine, and poly(aminoethyl methacrylate), or a pharmaceutically acceptable salt thereof.

[0802] It should be understood that the polymers described herein can be linear or branched. In some embodiments, the cationic polymer is linear. In some embodiments, the cationic polymer is a linear polymer selected from poly(ethyleneimine), poly(propyleneimine), polygluconine, polyallylamine, polyethyleneamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L-histidine, and poly(2-aminoethyl methacrylate). In some embodiments, the cationic polymer is a branched polymer selected from poly(ethyleneimine), poly(propyleneimine), polygluconine, polyallylamine, polyethyleneamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L-histidine, and poly(2-aminoethyl methacrylate).

[0803] In some embodiments, the cationic polymer comprises poly(ethyleneimine). In some embodiments, the poly(ethyleneimine) is a linear polymer. In some embodiments, the poly(ethyleneimine) is a branched polymer. In some embodiments, the poly(ethyleneimine) has an average molar mass of 1000 Da to 150000 Da, 5000 Da to 100000 Da, 10000 Da to 50000 Da, 15000 Da to 30000 Da, 20000 Da to 25000 Da, or about 22500 Da. In some embodiments, the poly(ethyleneimine) has an average molar mass of 22500 Da to 150000 Da.

[0804] In some embodiments, the functionalized particles of the present invention, particularly polymer complex (PLX) particles and esterified polymer complex (LPLX) particles, comprise polyamine derivatives (e.g., carboxylated polyamine derivatives) as particle-forming components. Polyamines form polycations in solution, which promote the formation of complexes with polyanions such as nucleic acids.

[0805] In some embodiments, the polyamine derivative which may be used herein as a delivery medium for polyanions comprises: a polyamine moiety comprising a plurality of amino groups; a plurality of carboxyl substituents comprising carboxyl groups bonded to the amino groups of the polyamine moiety via hydrophobic linkers; and a plurality of hydrophobic substituents bonded to the amino groups of the polyamine moiety.

[0806] In some embodiments, the polyamine derivative which may be used herein as a delivery medium for polyanions comprises: a polyamine moiety comprising a plurality of amino groups; a plurality of carboxyl substituents comprising carboxyl groups bonded to the amino groups of the polyamine moiety via hydrophobic linkers, wherein each of the carboxyl substituents comprises 6 to 40 carbon atoms, preferably 6 to 20 carbon atoms and more preferably 8 to 16 carbon atoms, and each of the hydrophobic linkers may comprise 1 to 3 heteroatoms selected from O, N and S; and a plurality of hydrophobic substituents bonded to the amino groups of the polyamine moiety, wherein each of the hydrophobic substituents comprises at least 2 carbon atoms, preferably 6 to 40 carbon atoms, and may comprise 1 to 3 heteroatoms selected from O, N and S, provided that the hydrophobic substituent has at least 6 carbon atoms.

[0807] In some embodiments, the polyamine derivative has a linear poly(ethyleneimine) moiety of 2 to 500 kDa (expressed as number average molecular weight), the carboxyl substituent has 10 to 16 carbon atoms and is an n-alkyl carboxylic acid, and the hydrophobic substituent has 1 to 12 carbon atoms and is alkyl, preferably n-alkyl and / or alkylarylalkyl.

[0808] In some embodiments, the polyamine derivative has a branched poly(ethyleneimine) moiety of 0.5 to 200 kDa (expressed as number average molecular weight), the carboxyl substituent has 10 to 16 carbon atoms and is an n-alkyl carboxylic acid, and the hydrophobic substituent has 1 to 12 carbon atoms and is alkyl, preferably n-alkyl and / or alkylarylalkyl.

[0809] In some embodiments, the particle-forming component includes a compound comprising the following formula:

[0810] .

[0811] The particles described herein, particularly polymer complex (PLX) and lipid polymer complex (LPLX) particles, may also contain polymers other than cationic polymers, i.e., non-cationic polymers and / or anionic polymers. Generally, anionic and neutral polymers are referred to herein as non-cationic polymers.

[0812] Anionic polymers

[0813] In some embodiments, the functionalized particles of this disclosure, particularly when in the form of a polymer complex (PLX), contain anionic polymers. In this specification, the term "anionic polymer" means a polymer as generally defined herein, which carries at least one anion, i.e., an ionic substance with a negative charge.

[0814] The anionic polymers described herein can be linear or branched and contain one or more anionic moieties or groups. In some embodiments, the anionic polymer is a polyanionic polymer, for example, a polymer having one or more anionic groups. In some embodiments, the anionic group is -CO2. - -OSO3 - 、or -OPO3 2- Group. In some embodiments, the anionic polymer is a homopolymer. In some embodiments, the anionic polymer is a heteropolymer.

[0815] In some embodiments, the anionic polymer is polyglutamic acid. In some embodiments, the anionic polymer is poly-L-glutamic acid. In some embodiments, the anionic polymer is polyaspartic acid. In some embodiments, the anionic polymer is poly-L-aspartic acid. In some embodiments, the anionic polymer is a polyphosphate.

[0816] In some embodiments, the anionic polymer is a homopolymer. In some embodiments, the anionic polymer is a homopolymer comprising about 10 to about 150 repeating monomer units. In some embodiments, the anionic polymer is a homopolymer comprising about 10 to about 100 repeating monomer units. In some embodiments, the anionic polymer is a homopolymer comprising about 20 to about 100 repeating monomer units. In some embodiments, the anionic polymer is a homopolymer comprising about 20 to about 80 repeating monomer units. In some embodiments, the anionic polymer is a homopolymer comprising about 50 repeating monomer units. In some embodiments, the anionic polymer is a homopolymer comprising about 100 repeating monomer units.

[0817] In some embodiments, the anionic polymer is a poly-L-glutamic acid homopolymer comprising about 10 to about 150 repeating units of glutamic acid. In some embodiments, the anionic polymer is a poly-L-glutamic acid homopolymer comprising about 10 to about 100 repeating units of glutamic acid. In some embodiments, the anionic polymer is a poly-L-glutamic acid homopolymer comprising about 20 to about 100 repeating units of glutamic acid. In some embodiments, the anionic polymer is a poly-L-glutamic acid homopolymer comprising about 20 to about 80 repeating units of glutamic acid. In some embodiments, the anionic polymer is a poly-L-glutamic acid homopolymer comprising about 50 repeating units of glutamic acid. In some embodiments, the anionic polymer is a poly-L-glutamic acid homopolymer comprising about 100 repeating units of glutamic acid.

[0818] Lipids and amphiphiles

[0819] The compositions of the present invention (such as aqueous dispersions, nucleic acid-lipid particles, and functionalized nucleic acid-lipid particles) also contain mixtures of lipids. The terms "lipid" and "lipid-like material" are broadly defined herein as molecules comprising one or more hydrophobic portions or groups and one or more hydrophilic portions or groups.

[0820] Lipids are generally insoluble or sparingly soluble in water, but soluble in many organic solvents. In an aqueous environment, their amphiphilic nature allows molecules to self-assemble into organized structures and different phases.

[0821] Lipids can contain polar and apolar (or non-polar) portions. As used herein, the term "amphiphilic" is broadly defined as a molecule containing both hydrophobic and hydrophilic portions and / or polar and non-polar portions. Both cationic and anionic lipids are amphiphilic because they both contain such groups. Therefore, in this specification, the terms "cationic lipid" and "cationic amphiphilic" are synonymous, and the terms "anionic lipid" and "anionic amphiphilic" are synonymous.

[0822] Hydrophobicity can be imparted by including nonpolar groups, including but not limited to long-chain saturated and unsaturated hydrocarbon groups (as defined and exemplified above), such as alkyl, alkenyl, and / or alkynyl groups, and groups substituted with one or more aryl, heteroaryl, or cycloalkyl groups (as defined and exemplified above). Hydrophilic groups may include polar and / or charged groups and include at least one amine and optional hydrophilic uncharged groups, such as hydroxyl, carbohydrate, mercapto, nitro, etc., and may also include anionic groups, such as phosphate, phosphonate, carboxylic acid, sulfate, sulfonate (all as defined and exemplified above), and other similar groups.

[0823] As used herein, the term "hydrophobic" with respect to a compound, group, or portion means that the compound, group, or portion is not attracted to water molecules and repels water molecules when present in an aqueous solution. In some embodiments, the term "hydrophobic" refers to any compound, group, or portion that is substantially immiscible or insoluble in an aqueous solution. In some embodiments, the hydrophobic compound, group, or portion is substantially nonpolar.

[0824] Examples of hydrophobic groups are hydrocarbon groups (as defined and exemplified above), such as alkyl, alkenyl, and / or alkynyl groups, and groups substituted with one or more aryl, heteroaryl, or cycloalkyl groups (as defined and exemplified above). Hydrophobic groups may have functional groups (e.g., ethers, thioethers, esters, dioxanes, halides, amides, sulfonamides, carbamates, etc.) and atoms other than carbon and hydrogen, provided that the group satisfies the condition of being substantially immiscible or insoluble in aqueous solutions.

[0825] The hydrophobic portion of a lipid can have 24 to 60 carbon atoms and can be a hydrocarbon group (typically containing alkyl, alkenyl, or alkynyl groups as described and exemplified above). The 24 to 60 carbon atoms can be segmented into two or more hydrophobic portions, each of which typically has at least 6 carbon atoms. An example of a segmented hydrophobic portion (where each segment is a hydrocarbon group) is a lipid containing a DCA portion as described in WO2011 / 003834, wherein each of the acyl or alkyl groups contains 12 to 20 carbon atoms. Another example is a lipid in which the hydrophobic portion contains a steroid portion (such as a cholesterol moiety).

[0826] The hydrophobic portion of the lipid preferably has 24 to 60 carbon atoms and may also be a heteroalkyl group, wherein the heteroatoms are selected from N, O, or S, forming one, two, three, or four uncharged groups such as ethers, thioethers, esters, amides, carbamates, sulfonamides, etc. The 24 to 60 carbon atoms may be segmented into two or more hydrophobic portions, provided that each such portion has at least 6 carbon atoms. Examples of segmented hydrophobic portions (where each segment is a hydrocarbon group) are lipids containing diacylglycerol or dialkylglycerol portions, wherein each of the acyl or alkyl groups contains 12 to 20 carbon atoms. Examples of hydrophobic portions (where each segment is a heteroalkyl group) are ester-branched portions of lipids, such as SM-102 or ALC-315, as defined and illustrated below.

[0827] Cationic lipids and cationic ionizable lipids

[0828] In some embodiments, the compositions of the present invention (such as aqueous dispersions, nucleic acid-lipid particles and functionalized nucleic acid-lipid particles) further contain cationic lipids or cationic ionizable lipids, or mixtures thereof.

[0829] As used herein, the term "cationic lipid" means a lipid or lipid-like material as defined herein that has a constitutive positive charge. In this context, "constitutive charge" means that the cationic lipid carries a positive charge at all physiological pH levels. Cationic lipids carrying a constitutively charged cationic moiety are typically salts of quaternary ammonium salts (as defined above) or organic bases (such as nitrogen-containing bases). Typically, such organic bases are strong bases (i.e., bases that are fully protonated when dissolved in a solvent (such as, but not limited to, aqueous solvents), such that the concentration of the unprotonated substance is too low to be measured).

[0830] In one embodiment, the cationic lipid is a monovalent cationic lipid.

[0831] In one embodiment, the cationic lipid contains a charged polar portion selected from the group consisting of guanidine, ammonium, imidazoline, pyridinium, amidine, and piperazine.

[0832] Examples of cationic lipids include, but are not limited to, 1,2-dialkoxy-3-dimethylammonium propane and 1,2-dienoxy-3-dimethylammonium propane (each alkyl or alkenyl moiety as defined and exemplified above and preferably having 12 to 20 carbon atoms), such as 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-diacyloxy-3-dimethylammonium propane (each acyl moiety having an alkyl or alkenyl moiety as defined and exemplified above and preferably having 12 to 20 carbon atoms), such as 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) or 1,2-dioleoyl-3-dimethylammonium-propane (D... ODAP); dimethyl dioctadecylammonium (DDAB); dioctadecyl dimethylammonium chloride (DODAC); 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazamonium (DMRIE); 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC); 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP); 1,2-dioleoyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE); and 2,3-dioleoyloxy-N-[2-(sperminecarbamate)ethyl]-N,N-dimethyl-1-propyltrifluoroacetate ammonium (DOSPA).

[0833] The structures of DODMA and DODAP are shown below.

[0834]

[0835] The structures of DOTMA, DOTAP and their analogues are shown below.

[0836]

[0837] The structures of DOTAP and other suitable homologues are shown below.

[0838]

[0839] The structures of DOTMA, DORIE, and other suitable homologues are shown below.

[0840]

[0841] Other suitable cationic lipids are described in Sun and Lu. Pharmaceutical Research , 2023, in https: / / doi.org / 10.1007 / s11095-022-03460-2.

[0842] In one embodiment, the lipid is a cationic ionizable lipid. As used herein, "cationic ionizable lipid" refers to a lipid or lipid-like material that, depending on whether it is protonated or deprotonated, has a net positive charge or is neutral, i.e., is not a permanently cationic lipid. Therefore, depending on the pH of the composition in which the cationic ionizable lipid is dissolved, the cationic ionizable lipid is either positively charged or neutral.

[0843] In some embodiments, the cationic ionizable lipid comprises a head group that includes at least one nitrogen atom (N) that is preferably protonated under physiological or slightly acidic conditions.

[0844] In one embodiment, the cationic lipid or cationic ionizable lipid is a compound represented by formula (TL-I):

[0845]

[0846] TL-I

[0847] Or its pharmaceutically acceptable salt, wherein:

[0848] L 1 and L 2 Each of the C1-Cs is independently and arbitrarily replaced. 30 Aliphatic groups;

[0849] L 3 It is a key, the C1-C that is optionally replaced. 10 Aliphatic groups, or optionally substituted 2- to 10-membered heteroaliphatic groups;

[0850] X 1and X 2 Each is independently selected from the bond, -OC(O)-, -C(O)O-, -S(O)2N(R) 1 )-、-N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-、-OC(S)C(R 1 )2-、-C(R 1 )2C(S)O- and –S-, where X 1 or X 2 One or two of them are selected from -S(O)2N(R) 1 )-、-N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-、-OC(S)C(R 1 )2-、-C(R 1 )2C(S)O- and –S-;

[0851] Each R 1 In each case, C1-C is independently and optionally substituted. 20 Aliphatic or H;

[0852] T 1 and T 2 Each of the C3-Cs is independently and arbitrarily replaced. 30 aliphatic;

[0853] G is -N(R) 2 )C(S)N(R 2 )2、-N + (R 3 )3、-OH、-N(R 2 )2、-N(R 5 )C(O)R 3 -N(R) 5 )S(O)2R 3 -N(R) 5 )C(O)N(R 3 )2、-CH(NR 2 ), or -R 4 ;

[0854] Each R 2 In each case, H, optionally substituted C1-C6 aliphatic or OR are independently selected. 3 A group consisting of two Rs; or two Rs 2 Together with the atoms to which they are attached, they form optionally substituted 4- to 12-membered heterocycles or optionally substituted 4- to 12-membered heteroaryl rings;

[0855] Each R 3 In each case, H and C1-C are independently chosen and optionally substituted. 10 Groups composed of aliphatic groups; and

[0856] R 4 It is a optionally substituted 4- to 12-membered heterocyclic compound, optionally substituted 4- to 12-membered heteroaryl compound, and –(CH2) 0-6 –OH or –(CH2) 0-6 –N(R 5 One or more substituted C6-C in )2 12 Aryl group, or oxidized to –(CH2). 0-6 –OH, or –(CH2) 0-6 –N(R 5 One or more substituted C3-C in )2 12 Cycloaliphatic;

[0857] Each R 5 Independently selected from H and optionally substituted C1-C6 aliphatic molecules.

[0858] In some implementations of formula (TL-I), L 1 and L 2 Each is independently –(CH2) 6-10 -

[0859] In some implementations of formula (TL-I), X 1 and X 2 Each is independently selected from -S(O)2N(R) 1 )-、-N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-、-OC(S)C(R 1 )2-、-C(R 1 )2C(S)O- and –S-.

[0860] In some implementations of formula (TL-I), X 1 and X 2 Each is -S(O)2N(R) 1 )-, where each R 1 R is independent 1 It is C1-C 10 Lipids.

[0861] In some implementations of formula (TL-I), T 1 and T 2 Each is independently selected from the arbitrarily replaced C3-C. 20 alkyl.

[0862] In some implementations of formula (TL-I), T 1 and T 2 Each is selected independently from:

[0863] , , , , , , , , , ;

[0864] , , , ;

[0865] , , and .

[0866] In some implementations of formula (TL-I), G is -N(R) 2 )C(S)N(R 2 )2 or -N(R 5 )S(O)2R 3 .

[0867] In some implementations of formula (TL-I), G is -N(H)C(S)N(R) 2 )2, where each R 2 Selected from optional substituted C1-C6 aliphatic and OH groups.

[0868] In some implementations of formula (TL-I), G is –OH.

[0869] In some implementations of formula (TL-I), G is selected from:

[0870] , , , , , , , , , and

[0871] In some implementations of formula (TL-I), -L 3 -G is selected from:

[0872] , , , , , , , , , and .

[0873] In some embodiments of formula (TL-I), the compound is represented by formula (TL-IIa):

[0874]

[0875] TL-IIa

[0876] Or its pharmaceutically acceptable salt.

[0877] In some embodiments of formula (TL-I), the compound is represented by formula (TL-IIc):

[0878]

[0879] TL-IIc

[0880] Or its pharmaceutically acceptable salt.

[0881] In some embodiments of formula (TL-I), the compound is represented by formula (TL-IIIb):

[0882]

[0883] (TL-IIIb)

[0884] Or its pharmaceutically acceptable salt.

[0885] In some embodiments of formula (TL-I), the compound is represented by formula (TL-IIIe):

[0886]

[0887] IIIe

[0888] or its pharmaceutically acceptable salt

[0889] In some embodiments of formula (TL-I), the compound is

[0890] In some embodiments of formula (TL-I), the compound is 7,7'-((4-hydroxybutyl)azanidinediyl)bis(N-hexyl-N-octylheptane-1-sulfonamide)

[0891] (BNT-51)

[0892] Or its pharmaceutically acceptable salt.

[0893] In some embodiments of formula (TL-I), the compound is 7,7'-((4-(3,3-dimethylthiourea)butyl)azanediyl)bis(N-hexyl-N-octylheptane-1-sulfonamide)

[0894] (BNT-52)

[0895] Or its pharmaceutically acceptable salt.

[0896] Thiolipid compounds of formula (TL-I) can be prepared according to the following general schemes 1, 2 and 3:

[0897]

[0898] General Scheme 1

[0899]

[0900] General Option 2

[0901]

[0902] General Scheme 3

[0903] The tail linker is any divalent linker, such as an aliphatic or heteroaliphatic group; the tail is a hydrophobic group (e.g., an aliphatic group); the head is a polar or cationic or ionizable head group; the tail linker is a biodegradable group (such as an ester) or a sulfur-containing moiety (e.g., a thioether, a sulfonyl group, or a sulfonamide); and the head-tail linker is the connection of one or more tails to the central atom or functional group (e.g., a tertiary amine group) of the head group.

[0904] Therefore, as described herein, this disclosure provides a method for preparing compounds represented by formula (TL-IV) or pharmaceutically acceptable salts thereof:

[0905]

[0906] TL-IV,

[0907] The method includes:

[0908] Compounds represented by formula (TL-V)

[0909]

[0910] TL-V

[0911] Compounds represented by one of the formulas (TLVIa)-(TL-VIc)

[0912]

[0913] (TL-VI)

[0914] and compounds represented by one of formulas (TL-VIIa)-(TL-VIIc)

[0915]

[0916] Contact in the presence of a reducing agent

[0917] in:

[0918] L 4 and L 5 Each of the C1-Cs in the equation is independently and optionally replaced. 30 Aliphatic groups;

[0919] L 6 It is a key, the C1-C that is optionally replaced. 10 Aliphatic groups, or optionally substituted 2- to 10-membered heteroaliphatic groups;

[0920] X 3 and X 4 Each is independently selected from the bond, -OC(O)-, -C(O)O-, -S(O)2N(R) 40 )-、-N(R 40 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 40 )2-、-OC(S)C(R 40 )2-、-C(R 40 )2C(S)O- or –S-, where X 3 or X 4 One or two of them are selected from -S(O)2N(R) 40 )-、-N(R 40 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 40 )2-、-OC(S)C(R 40 )2-、-C(R 40 )2C(S)O- or –S-;

[0921] Each R 40 In each case, C1-C is independently and optionally substituted. 20 Aliphatic or H;

[0922] T 3 and T 4Each of the C3-Cs is independently and arbitrarily replaced. 20 aliphatic;

[0923] G 1 It 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)2R 7 -N(R) 9 )C(O)N(R 7 )2、-CH(NR 7 ), or –R 8 ;

[0924] Each G 2 It can be either O or N2 independently;

[0925] Each G 3 It can be a halogen (e.g., Cl, Br, or I), -OTs, or OTf independently;

[0926] Each R 6 In each case, H, optionally substituted C1-C6 aliphatic or OR are independently selected. 7 A group consisting of two Rs; or two Rs 6 Together with the atoms to which they are attached, they form optionally substituted 4- to 12-membered heterocycles or optionally substituted 4- to 12-membered heteroaryl rings;

[0927] Each R 7 In each case, the group consisting of H and optionally substituted C1-C6 aliphatic molecules is selected independently;

[0928] R 8 It is a optionally substituted 4- to 12-membered heterocyclic compound, optionally substituted 4- to 12-membered heteroaryl compound, and –(CH2). 0-6 –OH or –(CH2) 0-6 –N(R 9 One or more substituted C6-C in )2 12 Aryl group, or oxidized to –(CH2). 0-6 –OH, or –(CH2) 0-6 –N(R 9 One or more substituted C3-C in )2 12 Cycloaliphatic; and

[0929] Each R 9 Independently selected from H and optionally substituted C1-C6 aliphatic molecules.

[0930] In some embodiments, the reducing agent is NaBH3CN or NaBH(OCOCH3)3. In some embodiments, the reducing agent is NaBH3CN. In some embodiments, the reducing agent is NaBH(OCOCH3)3.

[0931] In some embodiments, the method for preparing compounds as described herein further includes preparing compounds represented by formula (TL-VIa) or formula (TL-VIIa) in such a manner that G 1 It is O.

[0932]

[0933] To make the compounds represented by formula (TL-VIII) or the compounds represented by formula (TL-IX)

[0934]

[0935] Contact with oxidizing agents.

[0936] In some embodiments, the oxidant is DMSO, PCC, or DMP. In some embodiments, the oxidant is DMSO, and the method further includes reacting a compound represented by formula VIII or IX and DMSO with a sulfur trioxide pyridine complex (SO3). (Pyridine) contact.

[0937] In some embodiments, the method for preparing the compounds described herein further includes preparing compounds represented by formula (TL-VIII) or (TL-IX) by:

[0938]

[0939] To make the compound represented by formula (TL-X) or the compound represented by formula (TL-XI)

[0940]

[0941] With compounds represented by formula (TL-XII) or formula (TL-XIII):

[0942]

[0943] They contact in the presence of H2O2 and SOCl2; and each Y... 1 It is halogen.

[0944] In one embodiment, the cationic lipid or cationic ionizable lipid is selected from the group consisting of:

[0945] 7,7'-((4-hydroxybutyl)azanediyl)bis(N-hexyl-N-octylheptane-1-sulfonamide) (BNT-51);

[0946] 7,7'-((4-(3,3-dimethylthiourea)butyl)azanediyl)bis(N-hexyl-N-octylheptane-1-sulfonamide)

[0947] (BNT-52) ；

[0948] [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-315);

[0949] 1,2-Dioleoyloxy-3-dimethylaminopropane (DODMA);

[0950] 2,2-Dilinyl-4-dimethylaminoethyl-[1,3]-dioxane (DLin-KC2-DMA);

[0951] D-Lin-MC3-DMA (6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate).

[0952] 1,2-Dilinyloxy-N,N-dimethylaminopropane (DLin-DMA);

[0953] two(( Z )-Non-2-en-1-yl)-9-((4-(dimethylaminobutyryl)oxy)heptadecanedioate (L319);

[0954] Bis-(2-Butyloctyl)10-(N-(3-(dimethylamino)propyl)nonamido)-nonadecanedioate (A9);

[0955] (Heptadecanyl-9-yl-8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)octyl]amino}-octanoate)(L5);

[0956] Heptadecan-9-yl-8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}-octanoate)(SM-102);

[0957] O-[N-{(9Z,12Z)-octadec-9,12-dien-1-yl)}-N-{7-pentadecanylcarbonyloxyoctyl}-amino]4-(dimethylamino)butyrate (HY501);

[0958] 2-(two-((9) Z ,12Z )-Octadecano-9,12-dien-1-yl)amino)ethyl 4-(dimethylamino)butyrate (EA-2);

[0959] 4-((two-((9) Z ,12 Z )-Octadeca-9,12-dien-1-yl)amino)oxy)- N,N -Dimethyl-4-oxobutyl-4-amine (HYAM-2);

[0960] ((2-(4-(dimethylamino)butyryl)oxy)ethyl)azinediylbis(octane 8,1-diyl)bis(2-hexyldecanoate) (EA-405);

[0961] (2-(4-(dimethylamino)butyryl)oxy)azinediylbis(octane 8,1-diyl)bis(2-hexyldecanoate) (HY-405);

[0962] Palmitoyl-oleoyl-norarginine (PONA);

[0963] Guanidino-di[(heptadecyl)methyl]formic acid (GUADACA);

[0964] 4-Methylpyridinium-di(heptadecanyl)methylformic acid (MPDACA);

[0965] 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP);

[0966] 1,2-Dioleoyl-3-dimethylammonium propane (DODAP);

[0967] 1,2-Di-O-octadecenyl-3-trimethylammonium propane (DOTMA);

[0968] BODD-C2C4-PipZ (bis(2-octyldodecyl)3,3'-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate) ;

[0969] BHD-C2C2-PipZ (bis(2-hexyldecyl)3,3'-((4-(4-methylpiperazin-1-yl)butyl)azaalkyldiyl)dipropionate) ;

[0970] BODD-C2C2-DMA (bis(2-octyldodecyl)3,3'-((2-(dimethylamino)ethyl)azonyl)dipropionate) ;

[0971] BODD-C2C2-1Me-Pyr (bis(2-octyldodecyl)3,3'-((2-(1-methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate) ;

[0972] BODD-C2C2-Pyr (bis(2-octyldodecyl)3,3'-((2-(pyrrolidone-1-yl)ethyl)azanediyl)dipropionate) ;

[0973] Or a mixture of any of them.

[0974] In one embodiment, the cationic lipid is palmitoyl-oleoyl-norarginine (PONA). In one embodiment, the cationic lipid is 4-methylpyridinium-di(heptadecanyl)-methylformic acid (MPDACA). In one embodiment, the cationic lipid is 1,2-dioleoyloxy-3-trimethylammonium propane (DOTAP). In one embodiment, the cationic lipid is 1,2-dioleoyl-3-dimethylammonium-propane (DODAP).

[0975] In one embodiment, the cationic ionizable lipid is 7,7'-((4-hydroxybutyl)-azaalkyldiyl)bis(N-hexyl-N-octylheptane-1-sulfonamide) (BNT-51). In one embodiment, the cationic ionizable lipid is [(4-hydroxybutyl)azaalkyldiyl]-bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-315). In one embodiment, the cationic ionizable lipid is 1,2-dioleoyloxy-3-dimethylaminopropane (DODMA). In one embodiment, the cationic ionizable lipid is 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxacyclopentane (DLin-KC2-DMA). In one embodiment, the cationic ionizable lipid is heptadecano-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (D-Lin-MC3-DMA). In one embodiment, the cationic ionizable lipid is 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA). In another embodiment, the cationic ionizable lipid is di(( Z(L319)-Non-2-en-1-yl)-9-((4-(dimethylaminobutyryl)oxy)heptadecanedioate. In one embodiment, the cationic ionizable lipid is bis-(2-butyloctyl)10-(N-(3-(dimethylamino)propyl)-nonamido)-nonadecanedioate (A9). In one embodiment, the cationic ionizable lipid is (heptadecanedio-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)octyl]amino}-octanoate) (L5). In one embodiment, the cationic ionizable lipid is heptadecanedio-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}-octanoate) (SM-102). In one embodiment, the cationic ionizable lipid is O-[N-{(9Z,12Z)-octadec-9,12-dien-1-yl)}-N-{7-pentadecanylcarbonyloxyoctyl}-amino]4-(dimethylamino)butyrate (HY501). In one embodiment, the cationic ionizable lipid is 2-(di-((9Z,12Z)-octadec-9,12-dien-1-yl)}-N-{7-pentadecanylcarbonyloxyoctyl}-amino]4-(dimethylamino)butyrate (HY501). Z ,12 Z )-Octadecano-9,12-dien-1-yl)amino)ethyl 4-(dimethylamino)butyrate (EA-2).

[0976] In one embodiment, the cationic ionizable lipid is BODD-C2C4-PipZ. In one embodiment, the cationic ionizable lipid is BHD-C2C2-PipZ. In one embodiment, the cationic ionizable lipid is BODD-C2C2-DMA. In one embodiment, the cationic ionizable lipid is BODD-C2C2-1Me-Pyr. In one embodiment, the cationic ionizable lipid is BODD-C2C2-Pyr.

[0977] In some embodiments, the cationic ionizable lipids are selected from those generally and specifically described in WO 2018 / 087753.

[0978] In some implementations, the cationic ionizable lipids are selected from the group consisting of:

[0979]

[0980] In one embodiment, the cationic ionizable lipid is 4-((di-((9) Z ,12 Z )-Octadeca-9,12-dien-1-yl)amino)oxy)- N,N-Dimethyl-4-oxobut-4-amine (HYAM-2). In one embodiment, the cationic ionizable lipid is ((2-(4-(dimethylamino)butyryl)-oxy)ethyl)-azanediylbis(octane 8,1-diyl)bis(2-hexyldecanoate) (EA-405). In one embodiment, the cationic ionizable lipid is (2-(4-(dimethylamino)butyryl)-oxy)azanediylbis-(octane 8,1-diyl)bis(2-hexyldecanoate) (HY-405). In one embodiment, the cationic ionizable lipid is O-[N-{(9Z,12Z)-octadec-9,12-dien-1-yl)}-N-{7-pentadecanylcarbonyloxyoctyl}-amino]4-(dimethylamino)butanoate (HY501).

[0981] In one embodiment, the cationic lipid or cationic ionizable lipid is present in an amount of 20 mol% to 70 mol% of the total lipids present in the lipid mixture. In one embodiment, the cationic lipid or cationic ionizable lipid is present in an amount of 30 mol% to 60 mol% of the total lipids present in the lipid mixture. In one embodiment, the cationic lipid or cationic ionizable lipid is present in an amount of 40 mol% to 50 mol% of the total lipids present in the lipid mixture. In this context, the term "lipid mixture" applies to both aqueous dispersions and lipid mixture components of nucleic acid-lipid particles.

[0982] Other lipids

[0983] The lipid mixture in the compositions of the present invention (including aqueous dispersions, nucleic acid-lipid particles, and functionalized nucleic acid-lipid particles) may further comprise one or more additional lipids. In one embodiment, the one or more additional lipids comprise anionic amphiphiles, as defined and exemplified below. In one embodiment, the one or more additional lipids comprise neutral or zwitterionic lipids, as defined and exemplified below. In one embodiment, the one or more additional lipids comprise steroids, as defined and exemplified below. In one embodiment, the one or more additional lipids comprise neutral lipids, as defined and exemplified below. In one embodiment, the one or more additional lipids comprise neutral lipids (such as steroids), as defined and exemplified below.

[0984] neutral lipids

[0985] The compositions of the present invention (including aqueous dispersions, nucleic acid-lipid particles, and functionalized nucleic acid-lipid particles) may further comprise neutral lipids. Neutral lipids are preferably neutral phospholipids. In one embodiment, the phospholipids may be zwitterionic (i.e., carrying both a positive and a negative charge so that they are neutral at a pH range approximately neutral).

[0986] In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin. The hydrocarbon moiety of the acyl portion of the phospholipid is as defined above, but 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 portions of the phospholipid may be the same or different. In one embodiment, the acyl portion is a saturated fatty acid moiety having 8 to 24 carbon atoms (including the acyl carbon), preferably selected from the group consisting of tetracosyl, benzyl, arachidoyl, stearoyl, palmitoyl, myristoyl, lauroyl, decyl, and octyl moieties. In specific embodiments, the neutral phospholipid has a T value of 30°C or higher. m And it is selected from distearyl, dipalmitoyl, or stearoyl-palmitoyl moieties. In one embodiment, the acyl moieties are unsaturated fatty acid moieties having 14 to 22 carbon atoms (including acyl carbons), preferably selected from the group consisting of oleoyl, linoyl, and lineoyl moieties.

[0987] Examples of such phospholipids include diacylphosphatidylcholine, such as distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), bispentadecanylphosphatidylcholine, dilaurylphosphatidylcholine (DLPC), dipalmitoylphosphatidylcholine (DPPC), diarachidonicylphosphatidylcholine (DAPC), disamylphosphatidylcholine (DBPC), and ditrisacylphosphatidylcholine. DTPC, DLPC, POPC, 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholestylosuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-10-glycero-3-phosphocholine (C16) Lyso PC), and phosphatidylethanolamines, especially diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearate-phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), diphyranoylphosphatidylethanolamine (DPyPE), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phosphate-(1′-racemic-glycerol) (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), N-palmitoyl-D-erythrosine phosphocholine (SM), and other phosphatidylethanolamine lipids with different hydrophobic chains.

[0988] In some embodiments, the neutral lipids are selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DOPE and SM, or any mixture thereof.

[0989] Therefore, in some embodiments, the compositions described herein comprise a compound of formula (A) as defined herein, a cationic lipid or cationic ionizable lipid (as defined herein), and a phospholipid. In some embodiments, the compositions described herein comprise a compound of formula (A) as defined herein, a cationic lipid or cationic ionizable lipid, and a phospholipid selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DOPE, and SM, or mixtures thereof.

[0990] In one embodiment, neutral lipids are present in the lipid mixture at an amount of about 1 mol% to about 40 mol% of the total lipids present in the lipid mixture. In one embodiment, neutral lipids are present in the lipid mixture at an amount of about 2 mol% to about 25 mol% of the total lipids present in the lipid mixture. In one embodiment, neutral lipids are present in the lipid mixture at an amount of about 5 mol% to about 15 mol% of the total lipids present in the lipid mixture.

[0991] In one embodiment, 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 about 5 mol% to about 15 mol% of the total lipids present in the lipid mixture.

[0992] In one embodiment, the neutral lipid is 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 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 phosphatidylcholine and is present in the lipid mixture in an amount of about 5 mol% to about 15 mol% of the total lipids present in the lipid mixture.

[0993] In one embodiment, 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 about 5 mol% to about 15 mol% of the total lipids present in the lipid mixture.

[0994] In each of the above embodiments, in this context, the term "lipid mixture" applies to both aqueous dispersions and lipid mixture components of nucleic acid-lipid particles.

[0995] steroids

[0996] The compositions of the present invention (including aqueous dispersions, nucleic acid-lipid particles, and functionalized nucleic acid-lipid particles) may further comprise steroids. In one embodiment, the steroid comprises a sterol. In one embodiment, the steroid is cholesterol.

[0997] Therefore, in some embodiments, the lipid nanoparticle compositions described herein comprise cationic ionizable lipids (as defined herein) and cholesterol.

[0998] In one embodiment, 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.

[0999] In some embodiments, the combined concentration of neutral lipids (in particular, one or more phospholipids, especially phosphatidylcholine, such as DSPC) and steroids (in particular, cholesterol) may be from about 0 mol% to about 70 mol% of the total lipids present in the lipid mixture, such as from about 2 mol% to about 60 mol%, from about 5 mol% to about 55 mol%, or from about 5 mol% to about 50 mol%.

[1000] In each of the above embodiments, in this context, the term "lipid mixture" applies to lipid mixture components of both aqueous dispersions, nucleic acid-lipid particles, and functionalized nucleic acid-lipid particles.

[1001] Grafted lipids

[1002] The compositions described herein may also contain grafted lipids. In this specification, the term "grafted lipid" in its broadest sense means, as defined above (whether in the broadest or preferred sense), a lipid or lipid-like material conjugated to a polymer as defined above (whether in the broadest or preferred sense).

[1003] In one embodiment, the grafted lipid can act as a cloaked lipid. In this specification, the term "cloaked lipid" means a cloaked polymer (as defined above) conjugated to a lipid (as defined herein). In this specification, the term "cloaked polymer" means a polymer (as defined above) having the following characteristics: (a) a polar (hydrophilic) functional group; (b) a hydrogen bond acceptor group; (c) no hydrogen bond donor group; and (d) no net charge. In some embodiments, the cloaked polymer is designed to spatially stabilize the lipid particles by forming a protective hydrophilic layer that shields the hydrophobic lipid layer. In some embodiments, when such lipid particles are administered in vivo, the cloaked polymer can reduce their association with serum proteins and / or uptake generated by the reticuloendothelial system.

[1004] In one embodiment, the grafted lipid is a polyethylene glycol-conjugated lipid (also known as a PEG-lipid or PEGylated lipid). The term "PEGylated lipid" refers to a molecule comprising both a lipid moiety and a polyethylene glycol moiety. PEGylated lipids are known in the art. PEG-lipids may contain 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 sequential.

[1005] In some embodiments, the PEG-conjugated lipids (peg-modified lipids) are lipids having the following general formula:

[1006]

[1007] Or its pharmaceutically acceptable salt, tautomer or stereoisomer, wherein R 12 and R 13 Each of them independently is a straight-chain or branched alkyl or alkenyl chain containing 10 to 30 carbon atoms, wherein the alkyl / alkenyl chain is optionally interrupted by one or more ester bonds; and w has an average value in the range of 30 to 60.

[1008] In some implementations of this formula, R 12 and R 13 Each of them is independently a straight-chain alkyl chain containing 10 to 18 carbon atoms, preferably 12 to 16 carbon atoms.

[1009] In some implementations of this formula, R 12 and R 13 They are the same. In some implementations, R 12 and R 13 Each of them is a straight-chain alkyl chain containing 12 carbon atoms. In some embodiments, R 12 and R13 Each of them is a straight-chain alkyl chain containing 14 carbon atoms. In some embodiments, R 12 and R 13 Each of them is a straight-chain alkyl chain containing 16 carbon atoms.

[1010] In some implementations of this formula, R 12 and R 13 They are different. In some implementations, R 12 and R 13 One is a straight-chain alkyl chain containing 12 carbon atoms, and R 12 and R 13 Another one is a straight-chain alkyl chain containing 14 carbon atoms.

[1011] In some embodiments of this formula, w has an average value ranging from 40 to 50, such as an average value of 45.

[1012] In some embodiments of this formula, w is in the range of an average molecular weight of about 400 to about 6000 g / mol, such as about 1000 to about 5000 g / mol, about 1500 to about 4000 g / mol, or about 2000 to about 3000 g / mol. In some embodiments, R 12 and R 13 Each of them is a straight-chain alkyl chain containing 14 carbon atoms and w has an average value of 45.

[1013] Various PEG-conjugated lipids are known in the art and include, but are not limited to, peg-modified diacylglycerols (PEG-DAG) such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), peg-modified phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerols (PEG-S-DAG) such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG), peg-modified ceramides (PEG-cer), or PEG dialkoxypropyl carbamates such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl)carbamate or 2,3-di(tetradecanoyloxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate, etc.

[1014] In some embodiments, the PEG-conjugated lipid (peg-modified lipid) is or contains 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide. In some embodiments, the peg-modified lipid has the following structure:

[1015]

[1016] In some embodiments, the PEG-conjugated lipid (peg-modified lipid) is DMG-PEG 2000, for example, having the following structure:

[1017]

[1018] In some embodiments, the PEG-conjugated lipids (peg-modified lipids) have the following structure:

[1019]

[1020] Where n has an average value ranging from 30 to 60 (such as about 50). In some embodiments, the PEG-conjugated lipid (peg-modified lipid) is PEG. 2000 -C-DMA, which preferably refers to 3-N-[(ω-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxy-propylamine (MPEG-(2 kDa)-C-DMA) or methoxy-polyethylene glycol-2,3-bis(tetradecoxy)propylcarbamate (2000).

[1021] In some implementations, the nucleic acid particles described herein may comprise one or more PEG-conjugated lipids or peg-modified lipids as described in WO 2017 / 075531 and WO2018 / 081480, the entire contents of each of which are incorporated herein by reference for the purposes described herein.

[1022] Other examples of grafted lipids include lipids conjugated with poly(sarcosine) (pSar), lipids conjugated with poly(oxazoline) (POX), lipids conjugated with poly(oxazine) (POZ), lipids conjugated with poly(vinylpyrrolidone) (PVP), lipids conjugated with poly(N-(2-hydroxypropyl)-methacrylamide) (pHPMA), lipids conjugated with poly(dehydroalanine) (pDha), lipids conjugated with poly(aminoethoxyethoxyacetic acid) (pAEEA), and lipids conjugated with poly(2-methylaminoethoxyethoxyacetic acid) (pmAEEA).

[1023] In one embodiment, the grafted lipid is a polysarcosine-conjugated lipid, also referred to herein as a sarcosine-based lipid or pSar-lipid. The term "sarcosine-based lipid" refers to a molecule comprising both a lipid moiety and a polysarcosine (poly(N-methylglycine)) moiety, the polysarcosine moiety having repeating units as shown below:

[1024]

[1025] Where x refers to the number of sarcosine units. Polysarcosine may contain 2 to 200, 2 to 100, 5 to 200, 5 to 100, 10 to 200, 10 to 100, optionally 5 to 80, and preferably 10 to 70 sarcosine units.

[1026] In one embodiment, the grafted lipid is a lipid conjugated with polyoxazoline (POX) and / or polyoxazine (POZ) and / or POX / POZ, also referred to herein as a conjugate of POX and / or POZ polymers with one or more hydrophobic chains, or as oxazoline-based and / or oxazine-based lipids or POX and / or POZ-lipids. The terms "oxazoline-based lipid" or "POX-lipid" refer to a molecule comprising both a lipid moiety and a polyoxazoline moiety, the polyoxazoline moiety (pOx) having repeating units as shown below. The terms "oxazine-based lipid" or "POZ-lipid" refer to a molecule comprising both a lipid moiety and a polyoxazine moiety, the polyoxazine (pOz) moiety having repeating units as shown below. The terms "oxazoline- or oxazine-based lipids," "POX / POZ-lipids," or "POXZ-lipids" refer to molecules that comprise both a lipid moiety and a portion of a copolymer of polyoxazoline and polyoxazine (i.e., a polymer having repeating pOx and pOz units as shown below).

[1027]

[1028] And x refers to the number of pOx and / or pOz units. The total number of repeating pOx and / or pOz units in the polymer may include 2 to 200, 2 to 100, 5 to 200, 5 to 100, 10 to 200, 10 to 100, optionally 5 to 80, preferably 10 to 70 pOx and / or pOz units. [...

Claims

1. A functionalized particle comprising: (a) One or more particulate-forming components; (b) Nucleic acid payload; and (c) The portion that can bind to immunoglobulin D (IgD).

2. The functionalized particles of claim 1, comprising: (a) One or more particulate-forming components; (b) Nucleic acid payload; (c) A linker compound comprising: (i) A portion capable of incorporating the linker compound into the particles, and (ii) the first interaction part; and (d) Docking compounds, which include: (i) the second interaction component, and (ii) The portion that can bind to IgD; The first interacting portion and the second interacting portion can combine with each other.

3. The functionalized particles as described in claim 1 or claim 2, comprising: (a) One or more particulate-forming components; (b) Nucleic acid payload; (c) Linker compounds of formula (A): L‐X1‐P‐X2‐B1(A); and (d) Docking compounds of formula (I): B2-X3-B3(I) in: P either does not exist or contains a polymer; L includes a portion capable of incorporating the compound into the particles, which is attached to B1 when P is absent, or to the first end of the polymer P when P is present; B1 contains a portion that can bind to B2, wherein when P is absent, the portion B1 is attached to L, or when P is present, it is attached to the second end of the polymer P. X1 and X2 are either independent or connected parts; B2 contains the portion that can combine with B1; X3 is missing or is a connection part; and B3 contains a portion that can bind to IgD.

4. The functionalized particles according to any one of claims 1 to 3, wherein, The one or more particle-forming components comprise cationic lipids or cationic ionizable lipids, optionally wherein the functionalized particles are lipid nanoparticles (LNPs) or lipid complexes (LPXs).

5. The functionalized particles according to any one of claims 1 to 3, wherein, The one or more particle-forming components comprise a cationic polymer, wherein the functionalized particles are optionally polymeric composites (PLX) or esterified polymeric composites (LPLX).

6. The functionalized particles according to any one of claims 3 to 5, wherein: (a) L is a hydrophobic portion, optionally a lipid; or (b) The particles are polymer composites (PLX) and L is an anionic polymer.

7. The functionalized particles according to any one of claims 3 to 6, wherein, P is a polymer, optionally a hydrophilic polymer, optionally 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), or any mixture thereof).

8. The functionalized particles according to any one of claims 1 to 4, 6 or 7, wherein, The cationic lipid or cationic ionizable lipid is selected from the group consisting of: 7,7'-((4-hydroxybutyl)azanediyl)bis(N-hexyl-N-octylheptane-1-sulfonamide) (BNT-51); (BNT-52); [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-315); 1,2-Dioleoyloxy-3-dimethylaminopropane (DODMA); 2,2-Dilinyl-4-dimethylaminoethyl-[1,3]-dioxane (DLin-KC2-DMA); D-Lin-MC3-DMA (6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate). 1,2-Dilinyloxy-N,N-dimethylaminopropane (DLin-DMA); two(( Z )-Non-2-en-1-yl)-9-((4-(dimethylaminobutyryl)oxy)heptadecanedioate (L319); bis-(2-butyloctyl)10-(N-(3-(dimethylamino)propyl)nonamido)-nonadecanedioate (A9); (Heptadecanyl-9-yl-8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)octyl]amino}-octanoate)(L5); Heptadecan-9-yl-8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}-octanoate)(SM-102); O-[N-{(9Z,12Z)-octadec-9,12-dien-1-yl)}-N-{7-pentadecanylcarbonyloxyoctyl}-amino]4-(dimethylamino)butyrate (HY501); 2-(two-((9) Z ,12 Z )-Octadecano-9,12-dien-1-yl)amino)ethyl 4-(dimethylamino)butyrate (EA-2); 4-((two-((9) Z ,12 Z )-Octadeca-9,12-dien-1-yl)amino)oxy)- N,N -Dimethyl-4-oxobutyl-4-amine (HYAM-2); ((2-(4-(dimethylamino)butyryl)oxy)ethyl)azinediylbis(octane 8,1-diyl)bis(2-hexyldecanoate) (EA-405); (2-(4-(dimethylamino)butyryl)oxy)azinediylbis(octane 8,1-diyl)bis(2-hexyldecanoate) (HY-405); Palmitoyl-oleoyl-norarginine (PONA); Guanidino-di[(heptadecyl)methyl]formic acid (GUADACA); 4-Methylpyridinium-di(heptadecanyl)methylformic acid (MPDACA); 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP); 1,2-Dioleoyl-3-dimethylammonium propane (DODAP); and 1,2-Di-O-octadecenyl-3-trimethylammonium propane (DOTMA); BODD-C2C4-PipZ ; BHD-C2C2-PipZ ; BODD-C2C2-DMA ; BODD-C2C2-1Me-Pyr ; BODD-C2C2-Pyr ; Or a mixture of any of them.

9. The functionalized particles according to any one of claims 1 to 8, wherein, The particle-forming composition also includes one or more additional lipids, optionally The one or more additional lipids mentioned above are selected from: (a) Neutral or zwitterionic lipids, such as neutral or zwitterionic phospholipids, preferably wherein the neutral or zwitterionic phospholipids are selected from the group consisting of: Distearate phosphatidylcholine (DSPC); Dioleoylphosphatidylcholine (DOPC); Dimyristic phosphatidylcholine (DMPC); Dipalmitoylphosphatidylcholine (DPPC); Palmitoyl oleoyl-phosphatidylcholine (POPC); Dioleoylphosphatidylethanolamine (DOPE); 1,2-Di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPG); N-palmitoyl-D-erythrosine sphingocholine (SM); Or a mixture of any of them; (b) Steroids, preferably said steroids are cholesterol; and / or (c) Grafted lipids, preferably wherein the grafted lipids are selected from the group consisting of: poly(alkylene glycol) conjugated lipids, poly(sarcosine) conjugated lipids, poly(oxazoline) (POX) conjugated lipids; poly(oxazine) (POZ) conjugated lipids; poly(vinylpyrrolidone) (PVP) conjugated lipids; poly(N-(2-hydroxypropyl)-methacrylamide) (pHPMA) conjugated lipids; poly(dehydroalanine) (pDha) conjugated lipids; poly(aminoethoxyethoxyacetic acid) (pAEEA) conjugated lipids; and poly(2-methylaminoethoxyethoxyacetic acid) (pmAEEA) conjugated lipids; or mixtures thereof.

10. The functionalized particles according to any one of claims 3 to 9, wherein: (a) Part B1 of the compound of formula (A) comprises an antibody, an antibody-like molecule, or VHH, and part B2 of the compound of formula (I) is a peptide; or (b) Part B2 of the compound of formula (I) comprises an antibody, an antibody-like molecule, or a VHH, and part B1 of the compound of formula (A) is a peptide.

11. The functionalized particles according to any one of claims 3 to 10, wherein, Part B3 of the compound of formula (I) is a single-domain antibody (sdAb) capable of binding IgD, wherein the sdAb is optionally a variable heavy chain domain antibody (VHH), a heavy chain variable (VH) domain, or a variable neoantigen receptor antibody (VNAR), and preferably wherein the sdAb is VHH.

12. The functionalized particles according to any one of claims 3 to 11, wherein, Each of the portions B1 and B2 has a bonding domain comprising the following: (i) CDRs containing the following sequences: CDR1 – GVTISALNAMAMG (SEQ ID NO: 49) CDR2 – AVSERGNTY (SEQ ID NO: 53) CDR3 – LEDRVDSFHDY (SEQ ID NO: 51); or (ii) CDRs containing the following sequences: CDR1 – GVTISALNAMAMG (SEQ ID NO: 49) CDR2 – AVSERGNAM (SEQ ID NO: 50) CDR3 – LEDRVDSFHDY (SEQ ID NO: 51); or (iii) CDRs containing the following sequences: CDR1 – GVTISALNAMAMG (SEQ ID NO: 49) CDR2 – AVSSRGNAM (SEQ ID NO: 52) CDR3 – LEDRVDSFHDY (SEQ ID NO: 51); Optionally, one or more of the CDRs may contain one, two, or three amino acid mutations.

13. The functionalized particles according to any one of claims 3 to 12, wherein, X3 is a glycine-serine (GS) linker, such as a linker containing GGGGSGGGS (SEQ ID NO: 77).

14. The functionalized particles according to any of the preceding claims, wherein, The portion capable of binding with IgD includes: (i) Contains complementarity-determining regions (CDRs) of the following sequences: (A)CDR1 – GFTFEDYAIG (SEQ ID NO: 1) CDR2 – IRNRDGSTYYK (SEQ ID NO: 2) CDR3 – AALKLGRLLGLVHMPAQYEYDY (SEQ ID NO: 3); or (B)CDR1 – GFTFEDYA (SEQ ID NO: 82) CDR2 – IRNRDGST (SEQ ID NO: 83) CDR3 – AALKLGRLLGLVHMPAQYEYDY (SEQ ID NO: 3); or (C)CDR1 – DYAIG (SEQ ID NO: 105) CDR2 – AIRNRDGSTYYKDSVKG (SEQ ID NO: 106) CDR3-LKLGRLLGLVHMPAQYEYDY (SEQ ID NO: 107); (ii) CDRs containing the following sequences: (A)CDR1 – GFTFDDYAIG (SEQ ID NO: 4) CDR2 – ISIRNGKTYYS (SEQ ID NO: 7) CDR3 – AALKLGLGLVYLPAQYEYDY (SEQ ID NO: 8); or (B)CDR1 – GFTFDDYA (SEQ ID NO: 84) CDR2 – ISIRNGKT (SEQ ID NO: 86) CDR3 – AALKLGLGLVYLPAQYEYDY (SEQ ID NO: 8); or (C)CDR1 – DYAIG (SEQ ID NO: 105) CDR2 – CISIRNGKTYYSASVKG (SEQ ID NO: 110) CDR3 – LKLGGLGLVYLPAQYEYDY (SEQ ID NO: 111); or D) CDR1 – DYAIG (SEQ ID NO: 105) CDR2 – AISIRNGKTYYSASVKG (SEQ ID NO: 112) CDR3-LKLGGLGLVYLPAQYEYDY (SEQ ID NO: 111); (iii) CDRs containing the following sequences: (A)CDR1 – GFTFDDYAIG (SEQ ID NO: 4) CDR2 – IQNKDGSTYYK (SEQ ID NO: 5) CDR3 – AALKLGRLLRLVHMPAQYEYDY (SEQ ID NO: 6); or (B)CDR1 – GFTFDDYA (SEQ ID NO: 84) CDR2 – IQNKDGST (SEQ ID NO: 85) CDR3 – AALKLGRLLRLVHMPAQYEYDY (SEQ ID NO: 6); or (C)CDR1 – DYAIG (SEQ ID NO: 105) CDR2 – VIQNKDGSTYYKDSVKG (SEQ ID NO: 108) CDR3 – LKLGRLLRLVHMPAQYEYDY (SEQ ID NO: 109); (iv) CDRs containing the following sequences: (A)CDR1 – GSIFSLNTMG (SEQ ID NO: 9) CDR2 – SSWSGGSTYYA (SEQ ID NO: 10) CDR3 – AADRFTRLRAGADY (SEQ ID NO: 11); or (B)CDR1 – GSIFSLNT (SEQ ID NO: 87) CDR2 – SSWSGGST (SEQ ID NO: 88) CDR3 – AADRFTRLRAGADY (SEQ ID NO: 11); or (C)CDR1 – LNTMG (SEQ ID NO: 113) CDR2 – ASSWSGGSTYYADSVKE (SEQ ID NO: 114) CDR3-DRFTRLRAGADY (SEQ ID NO: 115); (v) CDRs containing the following sequences: (A)CDR1 – GFIFSNYAMY (SEQ ID NO: 12) CDR2 – INSDGGTNYK (SEQ ID NO: 13) CDR3 – AKGSTGYYSGGYDYES (SEQ ID NO: 14); or (B)CDR1 – GFIFSNYA (SEQ ID NO: 89) CDR2 – INSDGGT (SEQ ID NO: 90) CDR3 – AKGSTGYYSGGYD (SEQ ID NO: 91); or (C)CDR1 – NYAMY (SEQ ID NO: 116) CDR2 – RINSDGGTNYKDSVKG (SEQ ID NO: 117) CDR3-GSTGYYSGGYDYES (SEQ ID NO: 118); (vi) CDRs containing the following sequences: (A)CDR1 – RSTISNNPMG (SEQ ID NO: 15) CDR2 – ISRGGGTTNYA (SEQ ID NO: 16) CDR3 – NTNPWLSGS (SEQ ID NO: 17); or (B)CDR1 – RSTISNNP (SEQ ID NO: 92) CDR2 – ISRGGGTT (SEQ ID NO: 93) CDR3 – NTNPWLSGS (SEQ ID NO: 17); or (C)CDR1 – NNPMG (SEQ ID NO: 119) CDR2 – LISRRGGGTTNYADSVKG (SEQ ID NO: 120) CDR3-NPWLSGS (SEQ ID NO: 121); (vii) Contains CDRs with the following sequences: (A)CDR1 – GRTFSSAMA (SEQ ID NO: 18) CDR2 – ISASGTRRLYT (SEQ ID NO: 19) CDR3 – AADRVSIGGGIPDNAHVYPY (SEQ ID NO: 20); or (B)CDR1 – GRTFSSSA (SEQ ID NO: 94) CDR2 – ISASGTRR (SEQ ID NO: 95) CDR3 – AADRVSIGGGIPDNAHVYPY (SEQ ID NO: 20); or (C)CDR1 – SSAMA (SEQ ID NO: 122) CDR2 – GISASGTRRLYTDSAKG (SEQ ID NO: 123) CDR3 – DRVSIGGGIPDNAHVYPY (SEQ ID NO: 124); (viii) CDRs containing the following sequences: (A)CDR1 – GSIFSINTMG (SEQ ID NO: 21) CDR2 – FSKSGGSTYYA (SEQ ID NO: 22) CDR3 – AADRFTRLRAGADY (SEQ ID NO: 11); or (B)CDR1 – GSIFSINT (SEQ ID NO: 96) CDR2 – FSKSGGST (SEQ ID NO: 97) CDR3 – AADRFTRLRAGADY (SEQ ID NO: 11); or (C)CDR1 – INTMG (SEQ ID NO: 125) CDR2 – AFSKSGGSTYYADSVKG (SEQ ID NO: 126) CDR3-DRFTRLRAGADY (SEQ ID NO: 127); (ix) contains the following CDR sequences: (A)CDR1 – GSSFSINTMG (SEQ ID NO: 23) CDR2 – ISGSDGSTYYA (SEQ ID NO: 24) CDR3 – AADRFTRLRAGADY (SEQ ID NO: 11); or (B)CDR1 – GSSFSINT (SEQ ID NO: 98) CDR2 – ISGSDGST (SEQ ID NO: 99) CDR3 – AADRFTRLRAGADY (SEQ ID NO: 11); or (C)CDR1 – INTMG (SEQ ID NO: 125) CDR2 – AISGSDGSTYYADSVKG (SEQ ID NO: 128) CDR3-DRFTRLRAGADY (SEQ ID NO: 127); (x) contains the CDR of the following sequences: (A)CDR1 – GRTNSNYTIG (SEQ ID NO: 25) CDR2 – TSRSGRSTYNA (SEQ ID NO: 26) CDR3 – AATQWGASSLVGVYRY (SEQ ID NO: 28); or (B)CDR1 – GRTNSNYT (SEQ ID NO: 100) CDR2 – TSRSGRST (SEQ ID NO: 101) CDR3 – AATQWGASSLVGVYRY (SEQ ID NO: 28); or (C)CDR1 – NYTIG (SEQ ID NO: 129) CDR2 – ATSRSGRSTYNADSVKG (SEQ ID NO: 130) CDR3-TQWGASSLVGVYRY (SEQ ID NO: 27); (xi) contains the following CDRs: (A)CDR1 – GRTNSNYTIG (SEQ ID NO: 25) CDR2 – TSRSGRSTYNA (SEQ ID NO: 26) CDR3 – AATQWGASSLVGVYRY (SEQ ID NO: 28); or (B)CDR1 – GRTNSNYT (SEQ ID NO: 100) CDR2 – TSRSGRST (SEQ ID NO: 101) CDR3 – AATQWGASSLVGVYRY (SEQ ID NO: 28); or (C)CDR1 – NYTIG (SEQ ID NO: 129) CDR2 – ATSRSGRSTYNADSVKG (SEQ ID NO: 130) CDR3-TQWGASSLVGVYRY (SEQ ID NO: 27); (xii) contains the following CDRs: (A)CDR1 – GLIFSQYTMS (SEQ ID NO: 29) CDR2 – ITGSGGRILYE (SEQ ID NO: 30) CDR3 – AADRTLGIYDSRAHYNY (SEQ ID NO: 31); or (B)CDR1 – GLIFSQYT (SEQ ID NO: 102) CDR2 – ITGSGGRI (SEQ ID NO: 103) CDR3 – AADRTLGIYDSRAHYNY (SEQ ID NO: 31); or (C)CDR1 – QYTMS (SEQ ID NO: 131) CDR2 – GITGSGGRILYEDSLKG (SEQ ID NO: 132) CDR3-DRTLGIYDSRAHYNY (SEQ ID NO: 133); (xiii) CDRs containing the following sequences: (A)CDR1 – GRTLERYA (SEQ ID NO: 32) CDR2 – ITWNGNARYYE (SEQ ID NO: 33) CDR3 – AGGWRGMDDYNY (SEQ ID NO: 34); (B)CDR1 – GRTLERYA (SEQ ID NO: 32) CDR2 – ITWNGNAR (SEQ ID NO: 104) CDR3 – AGGWRGMDDYNY (SEQ ID NO: 34); or (C)CDR1 – RYA (SEQ ID NO: 134) CDR2 – AITWNGNARYYEDSVKG (SEQ ID NO: 135) CDR3-GWRGMDDYNY (SEQ ID NO: 136); Optionally, one or more of the CDRs may contain one, two, or three amino acid mutations.

15. A pharmaceutical composition comprising functionalized particles and a carrier according to any one of claims 1-14.

16. The functionalized particles according to any one of claims 1-14, used in methods for treating or diagnosing diseases, such as: (a) Targeted delivery of nucleic acid payloads to IgD-positive cells. (b) Targeted delivery of nucleic acid payloads to B cells, or (c) The nucleic acid payload described therein is a drug used to treat diseases such as HIV / AIDS, multiple myeloma, autoimmune diseases or allergic reactions.

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