Agents and methods for targeted delivery of nucleic acids to cells - Patents.com

Abstract

The present invention relates to agents and methods for targeted delivery of nucleic acids to cells. In some embodiments, the nucleic acid payload comprises a nucleic acid encoding an antigen receptor, such as a T cell receptor (TCR) or a chimeric antigen receptor (CAR). The agents and methods for targeted delivery of nucleic acids encoding antigen receptors described herein can be used to generate immune effector cells genetically modified to express antigen receptors in vitro / ex vivo or in vivo.

Description

[Technical Field]

[0001] The present invention relates to agents and methods for targeted delivery of nucleic acids to cells. The nucleic acid comprises DNA and / or RNA. In some embodiments, the nucleic acid comprises RNA, such as mRNA. In some embodiments, the nucleic acid encodes an antigen receptor, such as a T cell receptor (TCR) or a chimeric antigen receptor (CAR). Delivery of a nucleic acid encoding an antigen receptor, such as a TCR or a CAR, to a cell can be useful for generating immune effector cells genetically modified to express the antigen receptor. In some embodiments, the present invention involves particles, the particles comprising a hydrophobic moiety (targeting compound) having a binding moiety covalently attached thereto, and the particles carrying a nucleic acid payload, e.g., a nucleic acid encoding an antigen receptor. Cell targeting can be achieved by direct or indirect binding of the targeting compound to a cell surface antigen on the target cell of interest.

[0002] In some embodiments, the binding moiety of the targeting compound binds to a cell surface antigen on a target cell, e.g., an immune effector cell, to target the particle having a nucleic acid payload to the target cell. In these embodiments, the binding moiety of the targeting compound can be a construct with affinity for a cell surface target, e.g., a membrane protein, and can include an antibody or antibody fragment.

[0003] In some embodiments, the binding moiety of the targeting compound binds to a compound (docking compound) that binds to a cell surface antigen on a target cell, such as an immune effector cell, to target a particle carrying a nucleic acid payload to the target cell. In some embodiments, the docking compound comprises a peptide or polypeptide. In some embodiments, the docking compound comprises a binding moiety that binds to the target cell (primary targeting moiety) and an additional binding moiety that binds to the binding moiety of the targeting compound. The binding moiety of the targeting compound can bind to the binding moiety on the docking compound, and the primary targeting moiety can then bind to a target antigen on the target cell, such as an antigen on an immune effector cell, thereby precisely delivering the nucleic acid payload to the target cell, such as an immune effector cell.

[0004] In many areas of medical therapy and diagnostics, it is desirable to selectively deliver agents, such as nucleic acids, to specific cells within the body of a subject, such as a patient.

[0005] The present invention relates to an approach in which particles containing a nucleic acid payload and a targeting compound are used. The targeting compound comprises (i) a hydrophobic portion for incorporation into the particle and (ii) a binding portion covalently attached to the hydrophobic portion for direct or indirect targeting of the particle to a target cell and delivery of the nucleic acid payload to the target cell. Cell targeting can be achieved by direct or indirect binding of the targeting compound to a cell surface antigen on the target cell of interest.

[0006] In some embodiments, particles containing a nucleic acid payload and a targeting compound are administered. In some embodiments, the binding moiety of the targeting compound binds to target cells, for example, by binding to a cell surface antigen, thereby causing the nucleic acid payload to be internalized in cells. A common example of the target cell binding moiety on the targeting compound is an antibody.

[0007] In some embodiments, particles containing a nucleic acid payload and a targeting compound are administered, along with a docking compound that binds to target cells, for example, by binding to a cell surface antigen. In these embodiments, the targeting compound may comprise a binding moiety that targets a moiety on the docking compound. In some embodiments, a docking compound bound to a particle containing a nucleic acid payload, for example, a nucleic acid encoding an antigen receptor, via a targeting compound is administered. The docking compound may bind to target cells, for example, by binding to a cell surface antigen, thereby resulting in intracellular uptake of the nucleic acid payload. A common example of a pair of interacting moieties on the targeting compound and the docking compound is an antibody / antigen system. A common example of a target cell binding moiety on the docking compound is an antibody. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention relates to agents and methods for targeted delivery of nucleic acid payloads to cells. In some embodiments, the nucleic acid payload comprises a nucleic acid encoding an antigen receptor, such as a T cell receptor (TCR) or a chimeric antigen receptor (CAR). The agents and methods for targeted delivery of nucleic acids encoding antigen receptors described herein can be used to generate immune effector cells genetically modified to express the antigen receptor in vitro / ex vivo or in vivo. Genetic modification is achieved using particles described herein that contain a nucleic acid encoding the antigen receptor for genetic modification and a targeting compound for targeting the immune effector cells, either directly or via a docking compound that binds to the targeting compound. The particles can deliver nucleic acids to cells in vitro / ex vivo as well as in vivo. Immune effector cells genetically modified to express the antigen receptor described herein are useful in treating diseases where targeting cells, such as diseased cells that express antigens, such as tumor antigens, is beneficial. Target cells can express antigens on the cell surface for recognition by CARs or in the context of MHC for recognition by TCRs. The treatments described herein can provide selective eradication of such cells that express an antigen, thereby minimizing adverse effects on normal cells that do not express the antigen. Immune effector cells genetically modified to express an antigen receptor, e.g., a CAR or TCR, that targets cells via binding to the antigen (or its processing product) are provided to a subject, such as by administering the genetically modified immune effector cells to the subject or generating genetically modified immune effector cells in the subject. In some embodiments, the immune effector cells are CD3+ T cells. In some embodiments, the target cell binding moiety (which may be present on the targeting compound or docking compound described herein) binds to the CD3 receptor on T cells. In some embodiments, the immune effector cells are CD8+ T cells. In some embodiments, the target cell binding moiety binds to the CD8 receptor on T cells. In some embodiments, the immune effector cells are CD4+ T cells. In some embodiments, the target cell binding moiety binds to the CD4 receptor on T cells.The methods and agents described herein and immune effector cells genetically modified to express antigen receptors are particularly useful for treating diseases characterized by disease cells expressing antigens to which immune effector cells are directed. In some embodiments, the immune effector cells have binding specificity for the disease-associated antigen when present on disease cells via a CAR. In some embodiments, the immune effector cells have binding specificity for the processed product of the disease-associated antigen when presented on disease cells via a TCR. In some embodiments, the cells are genetically modified to stably express the antigen receptor on their surface. In some embodiments, the cells are genetically modified to transiently express the antigen receptor on their surface. [Means for solving the problem]

[0009] In one aspect, the present invention provides a compound of formula: L-X1-P-X2-B (In the formula, P comprises a polymer; L comprises a hydrophobic moiety attached to the polymer; B comprises a moiety attached to the polymer selected from the group consisting of a moiety that binds to a cell surface antigen, a peptide tag, and a moiety that binds to a peptide tag; X1 is absent or a first linking moiety; and X2 is absent or a second linking moiety The present invention relates to the compound

[0010] This compound is also referred to herein as a "targeting compound."

[0011] In some embodiments, the hydrophobic moiety comprises a moiety selected from vitamin E, a dialkylamine, a diacylglyceride, and a ceramide.

[0012] In some embodiments, the hydrophobic moiety comprises two C8-C24 hydrocarbon chains.

[0013] In some embodiments, the hydrophobic moiety comprises a lipid.

[0014] In some embodiments, the hydrophobic moiety comprises a phospholipid.

[0015] In some embodiments, the hydrophobic moiety comprises a moiety selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine), and mixtures thereof.

[0016] In some embodiments, the polymer is a hydrophilic polymer.

[0017] 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), derivatives and combinations thereof.

[0018] In some embodiments, X2 comprises the reaction product of a thiol or cysteine ​​reactive group with a thiol or cysteine ​​group of a compound comprising moiety B.

[0019] In some embodiments, the thiol or cysteine ​​reactive group comprises a maleimide group.

[0020] In some embodiments, the compound comprises the reaction product of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)] and a compound comprising the formula SH(CH2)nC(O)-B, where n is in the range of 1 to 5, preferably n is 2.

[0021] In some embodiments, the compound has the formula [ka] is a compound of

[0022] In some embodiments, B comprises a peptide or polypeptide.

[0023] In some embodiments, the moiety that binds to a cell surface antigen or peptide tag comprises an antibody or antibody-like molecule.

[0024] In some embodiments, the antibody-like molecule comprises an antibody fragment or a DARPin.

[0025] In some embodiments, the cell surface antigen is characteristic of an immune effector cell.

[0026] In some embodiments, the cell surface antigen is selected from the group consisting of CD4, CD8 and CD3.

[0027] In some embodiments, the peptide tag comprises an ALFA tag.

[0028] In one aspect, the present invention relates to functionalized particles comprising one or more particle-forming components, a nucleic acid payload, and a compound (targeting compound) as described above, wherein the compound is incorporated into the particle via a hydrophobic moiety.

[0029] In some embodiments, B comprises a moiety that binds to a cell surface antigen.

[0030] In some embodiments, B comprises a peptide tag or a moiety that binds to a peptide tag, and the functionalized particle has the formula: B'-X3-B'' (In the formula, B' comprises a moiety that binds to B; X3 is absent or a linking moiety; and B'' contains a portion that binds to a cell surface antigen) The compound further includes the compound of formula (I).

[0031] Compounds of formula B'-X3-B'' are also referred to herein as "docking compounds."

[0032] In some embodiments, the compound of formula B'-X3-B'' comprises a peptide or polypeptide.

[0033] In some embodiments, the moiety that binds to a cell surface antigen comprises an antibody or antibody-like molecule.

[0034] In some embodiments, the antibody-like molecule comprises an antibody fragment or a DARPin.

[0035] In some embodiments, the cell surface antigen is characteristic of an immune effector cell.

[0036] In some embodiments, the cell surface antigen is selected from the group consisting of CD4, CD8 and CD3.

[0037] In some embodiments, B comprises a peptide tag and B' comprises a moiety that binds to the peptide tag.

[0038] In some embodiments, B' comprises a peptide tag and B comprises a moiety that binds to the peptide tag.

[0039] In some embodiments, the moiety that binds to the peptide tag comprises an antibody or antibody-like molecule.

[0040] In some embodiments, the antibody-like molecule comprises an antibody fragment or a DARPin.

[0041] In some embodiments, the peptide tag comprises an ALFA tag.

[0042] In some embodiments, the peptide tag comprises an ALFA tag, and the moiety that binds to the peptide tag comprises a VHH domain comprising the CDR1 sequence VTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY.

[0043] In some embodiments, the particles are lipid particles, polymer particles, or mixtures thereof.

[0044] In some embodiments, the particle is a non-viral particle.

[0045] In some embodiments, the particles are nanoparticles.

[0046] In some embodiments, the nucleic acid comprises DNA and / or RNA.

[0047] In some embodiments, the nucleic acid comprises a nucleic acid encoding an antigen receptor.

[0048] In some embodiments, the antigen receptor comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0049] In one aspect, the present invention relates to a method for delivering nucleic acids to cells expressing a cell surface antigen, the method comprising adding to the cells a composition comprising the functionalized particles described above.

[0050] In some embodiments, B or B'' comprises a moiety that binds to a cell surface antigen.

[0051] In some embodiments, the cells are ex vivo.

[0052] In some embodiments, the cell is present in a subject and the method comprises administering the composition to the subject.

[0053] In some embodiments, the cells comprise immune effector cells.

[0054] In some embodiments, the immune effector cells comprise T cells.

[0055] In some embodiments, the immune effector cells comprise CD8+ and / or CD4+ T cells.

[0056] In some embodiments, the cell surface antigen is characteristic of an immune effector cell.

[0057] In some embodiments, the cell surface antigen is selected from the group consisting of CD4, CD8 and CD3.

[0058] In some embodiments, the nucleic acid comprises a nucleic acid encoding an antigen receptor.

[0059] In some embodiments, the antigen receptor comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0060] In some embodiments, the method is a method for preparing a genetically modified cell.

[0061] In some embodiments, the method is a method of preparing immune effector cells genetically modified to express an antigen receptor.

[0062] In some embodiments, the genetic modification is transient or stable.

[0063] In some embodiments, the genetic modification is carried out by a viral-based method, a transposon-based method, or a gene editing-based method.

[0064] In one aspect, the present invention relates to a method for preparing immune effector cells genetically modified to express an antigen receptor, the method comprising adding to the immune effector cells a composition comprising the functionalized particles described above, wherein the nucleic acid comprises a nucleic acid encoding the antigen receptor.

[0065] In some embodiments, B or B'' comprises a moiety that binds to a cell surface antigen on an immune effector cell.

[0066] In some embodiments, the immune effector cells are present ex vivo.

[0067] In some embodiments, the immune effector cells are present in a subject and the method comprises administering the composition to the subject.

[0068] In some embodiments, the immune effector cells comprise T cells.

[0069] In some embodiments, the immune effector cells comprise CD8+ and / or CD4+ T cells.

[0070] In some embodiments, the cell surface antigen is characteristic of an immune effector cell.

[0071] In some embodiments, the cell surface antigen is selected from the group consisting of CD4, CD8 and CD3.

[0072] In some embodiments, the antigen receptor comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0073] In some embodiments, the genetic modification is transient or stable.

[0074] In some embodiments, the genetic modification is carried out by a viral-based method, a transposon-based method, or a gene editing-based method.

[0075] In one aspect, the present invention provides a method of treating a subject, comprising: (I) preparing ex vivo immune effector cells genetically modified to express an antigen receptor using the method described above; and (II) administering immune effector cells genetically modified to express an antigen receptor to a subject. The present invention relates to a method comprising:

[0076] In one aspect, the present invention relates to a method of treating a subject, comprising preparing in vivo immune effector cells genetically modified to express an antigen receptor using the methods described above. [Brief explanation of the drawings]

[0077] [Figure 1] Preparation and Characterization of RNA-Containing Functionalized Lipid Nanoparticles (LNPs). LNPs were formulated with varying mole percent Alfa-tagged lipids (lipid mixture: DODMA / Chol / DOPE / C16-PEG-Ceramide / DSPE-PEG2k-Alfa 40 / 48 / 10 / 2-x / x = 0, 0.2, 0.5, 1; N / P ratio: 4; cargo: Thy1.1 / Luc RNA 1:1 w / w; RNA concentration: 0.1 μg / μl). aCD8-DARPin x NbAlfa was added via post-functionalization [w / w* = ligand-to-cargo ratio 0.55]. Successful RNA incorporation was verified via agarose gel electrophoresis. Size and PDI were measured via dynamic light scattering at an RNA concentration of 0.005 g / L for LNPs with or without ligand. **PDI not measurable. [Figure 2A](Figure 2) T cell targeting using LNPs decorated with aCD3 ligand. LNPs were formulated with or without ligand (cargo: Thy1.1 / Luc RNA 1:1 w / w; N / P ratio: 4; lipid mixture: DODMA / cholesterol / DOPE / C16 PEG2k-ceramide / DSPE-PEG2k-Alfa [40 / 48 / 10 / 1.8 / 0.2]; aCD3-VHH X NbAlfa post-functionalization [w / w* = ligand-to-cargo ratio 1.15]; RNA concentration: 0.1 μg / μl). Successful RNA incorporation was verified via agarose gel electrophoresis. For transfection studies, 15 μl of each formulation was prediluted in 50 μl X-Vivo 15 in an ultra-low attachment 96-well plate. 1e6 thawed human PBMCs were diluted in 50 μl X-Vivo 15 (containing 10% PHS) and added to the nanoparticle diluent (1500 ng RNA). After 30 min of incubation (37°C, 5% CO2), 200 μl of X-Vivo 15 + 5% PHS was added per well, and the cells were cultured for an additional 18 h (37°C, 5% CO2). Cell type-specific transfection (Thy1.1) and T cell activation (CD69) were analyzed via flow cytometry. (A) Characterization of LNPs. Diameter and PDI were determined via DLS measurements. [Figure 2B] (B) Depicts cell type-specific Thy1.1 (transfection, y axis) signals in viable CD14+ monocytes, CD56+ NK cells, CD19+ B cells, CD3+ T cells, CD4+ T cells, or CD8+ T cells. [Figure 2C] (C) Histogram showing CD69 activation marker expression on T cell populations within treated PBMCs. [Figure 3]T cell targeting using LPLX decorated with aCD8 DARPin. LPLX was functionalized with two different ligands: aCD8 DARPin X Spycatcher or aCD8 DARPin X NbAlfa. The LPLX contained cationic Viromer L3 polymer and functionalized lipids for RNA encapsulation (cargo: Thy1.1 / Luc RNA 1:1 w / w; RNA concentration: 0.1 μg / μL; N / P ratio: 7.5; ligand-to-cargo w / w ratio: 0.25 for aCD8 DARPin X Spycatcher and 0.1 for aCD8 DARPin X NbAlfa; ligand-to-functionalized lipid molar ratio: 1.5:1 for aCD8 DARPin X Spycatcher / Spytag PEG2k-DSPE and 3.5:1 for aCD8 DARPin X NbAlfa / Alfatag PEG2k-DSPE). Diameter and PDI were determined via DLS measurements. Successful RNA integration was verified via agarose gel electrophoresis. For transfection studies, 0.5 or 0.1 μl of each formulation was prediluted in 50 μl X-Vivo 15 in a 96-well plate. 16 thawed human T cells were diluted in 50 μl X-Vivo 15 and added to the nanoparticle dilution (50 or 100 ng RNA). After 30 min of incubation (37°C, 5% CO2), 200 μl of X-Vivo 15 + 5% PHS was added per well, and the cells were cultured for an additional 18 h (37°C, 5% CO2). CD4- and CD8-specific transfection (Thy1.1) was analyzed via flow cytometry below. Thy1.1 (transfection, y-axis) signals in viable CD4+ or CD8+ T cells are depicted. [Figure 4A](Figure 4) T cell targeting using LPLX decorated with different ligands. LPLX was functionalized using a fusion protein composed of a CD8-targeting ligand fused to X NbAlfa. The CD8-targeting domain was either a DARPin or a VHH nanobody. One DARPin and four different nanobodies were tested. LPLX contained Viromer L3 for RNA encapsulation and a DSPE-PEG2k alpha tag for ligand binding (cargo: Thy1.1 / Luc RNA 1:1 w / w; RNA concentration: 0.1 μg / μL; N / P ratio: 7.5; ligand-to-cargo w / w ratios of 0.05, 0.1, and 0.2 for aCD8 VHH X NbAlfa; ligand-to-cargo w / w ratio of 0.2 for aCD8 DARPin X NbAlfa; molar ratio of ligand to functionalized lipid: 3.5:1). [Figure 4B] (Figure 4 (continued)) Diameter and PDI were determined via DLS measurements. Successful RNA incorporation was verified via agarose gel electrophoresis. For transfection studies, 0.1 μl of each formulation was prediluted in 50 μl X-Vivo 15 in a 96-well plate. 2e5 thawed human T cells were diluted in 50 μl X-Vivo 15 and added to the nanoparticle dilution (100 ng RNA). After 30 min of incubation (37°C, 5% CO2), 200 μl of X-Vivo 15 + 5% PHS was added per well, and the cells were cultured for an additional 18 h (37°C, 5% CO2). CD4- and CD8-specific transfection (Thy1.1) was analyzed via flow cytometry below. The center graph shows particle condition (see table below)-specific CD8+ T cell transfection efficiency, calculated by multiplying the transfected (Thy1.1+) percentage of CD8+ T cells by the MFI of the Thy1.1+ CD8+ cell population. Dot blots corresponding to the conditions in the graph markers with boxes are depicted in the upper part of the figure. These blots show Thy1.1 signal in viable CD8+ T cells (upper blot) or viable CD4+ T cells (lower blot). [Figure 5A](Figure 5) T cell targeting using LPLX decorated with different ligands. LPLX was functionalized with fusion proteins composed of CD8, CD4, or CD3 targeting ligands fused to XNbAlfa. The targeting domains were either DARPin, VHH nanobody, or scFv. One DARPin, nine different nanobodies, and two different scFvs were tested. LPLX contains Viromer L3 for RNA encapsulation and a DSPE-PEG2k alpha tag for ligand binding (cargo: Thy1.1 / Luc RNA 1:1 w / w; RNA concentration: 0.1 μg / μL; N / P ratio: 7.5; ligand-to-cargo w / w ratios of 0.05, 0.1, and 0.2 for aCD4 VHH × NbAlfa and aCD3 scFv × NbAlfa; ligand-to-cargo w / w ratio of 0.2 for aCD8 DARPin × NbAlfa and aCD3 VHH × NbAlfa; molar ratio of ligand to functionalized lipid of 3.5:1). [Figure 5B] (Figure 5 (continued)) LPLX was functionalized with fusion proteins composed of CD8, CD4, or CD3 targeting ligands fused to X NbAlfa. The targeting domains are either DARPins, VHH nanobodies, or scFvs. One DARPin, nine different nanobodies, and two different scFvs were tested. LPLX contains Viromer L3 for RNA encapsulation and a DSPE-PEG2k alpha tag for ligand binding (cargo: Thy1.1 / Luc RNA 1:1 w / w; RNA concentration: 0.1 μg / μL; N / P ratio: 7.5; ligand-to-cargo w / w ratios of 0.05, 0.1, and 0.2 for aCD4 VHH × NbAlfa and aCD3 scFv × NbAlfa; ligand-to-cargo w / w ratio of 0.2 for aCD8 DARPin × NbAlfa and aCD3 VHH × NbAlfa; molar ratio of ligand to functionalized lipid of 3.5:1). [Figure 5C](Figure 5 (continued)) Diameter and PDI were determined via DLS measurements. Successful RNA incorporation was verified via agarose gel electrophoresis. For transfection studies, 0.1 μl of each formulation was prediluted in 50 μl X-Vivo 15 in a 96-well plate. 2e5 thawed human T cells were diluted in 50 μl X-Vivo 15 and added to the nanoparticle dilution (100 ng RNA). After 30 min of incubation (37°C, 5% CO2), 200 μl of X-Vivo 15 + 5% PHS was added per well, and the cells were cultured for an additional 18 h (37°C, 5% CO2). CD4- and CD8-specific transfection (Thy1.1) was analyzed via flow cytometry below. The center graph shows particle condition (see table below)-specific CD8+ (left y-axis; dark gray bars) and CD4+ (right y-axis; light gray bars) T cell transfection efficiencies. Values ​​were calculated by multiplying the transfected (Thy1.1+) percentage of CD8 or CD4+ T cells by the MFI of the respective Thy1.1+ cell population. Dot blots corresponding to the conditions in the graph markers with boxes are depicted in the upper part of the figure. These blots show Thy1.1 signals in viable CD8+ T cells (upper blot) or viable CD4+ T cells (lower blot). [Figure 5D](Figure 5 (continued)) Diameter and PDI were determined via DLS measurements. Successful RNA incorporation was verified via agarose gel electrophoresis. For transfection studies, 0.1 μl of each formulation was prediluted in 50 μl X-Vivo 15 in a 96-well plate. 2e5 thawed human T cells were diluted in 50 μl X-Vivo 15 and added to the nanoparticle dilution (100 ng RNA). After 30 min of incubation (37°C, 5% CO2), 200 μl of X-Vivo 15 + 5% PHS was added per well, and the cells were cultured for an additional 18 h (37°C, 5% CO2). CD4- and CD8-specific transfection (Thy1.1) was analyzed via flow cytometry below. The center graph shows particle condition (see table below)-specific CD8+ (left y-axis; dark gray bars) and CD4+ (right y-axis; light gray bars) T cell transfection efficiencies. Values ​​were calculated by multiplying the transfected (Thy1.1+) percentage of CD8 or CD4+ T cells by the MFI of the respective Thy1.1+ cell population. Dot blots corresponding to the conditions in the graph markers with boxes are depicted in the upper part of the figure. These blots show Thy1.1 signals in viable CD8+ T cells (upper blot) or viable CD4+ T cells (lower blot). [Figure 6]T Cell Targeting Using LNPs with Different Lipid Compositions. LNPs with different lipid compositions and aCD3 VHH x NbAlfa ligands were formulated (cargo: Thy1.1 / Luc RNA 1:1 w / w; N / P ratio: 4; lipid mixture: ionizable lipid / cholesterol / DOPE / C16 PEG2k-ceramide / DSPE-PEG2k-Alfa [40 / 48 / 10 / 1.8 / 0.2], with DODMA (C12), HY-501 (H12), or EA-405 (E12) as ionizable lipids; aCD3-VHH x NbAlfa post-functionalization [w / w* = ligand-to-cargo ratio 1.15; ligand-to-functionalized lipid molar ratio 1.5:1]; RNA concentration: 0.1 μg / μl). All LNPs had a diameter of less than 120 nm and a PDI of less than 0.2, as determined via DLS measurements. Successful RNA integration was verified via agarose gel electrophoresis. For transfection studies, 5 or 15 μl (500 ng or 1500 ng doses) of each formulation was prediluted in 50 μl X-Vivo 15 in an ultra-low attachment 96-well plate. 16 thawed human PBMCs were diluted in 50 μl X-Vivo 15 (containing 10% PHS) and added to the nanoparticle dilutions. After 30 min of incubation (37°C, 5% CO2), 200 μl of X-Vivo 15 + 5% PHS was added per well, and the cells were cultured for an additional 18 h (37°C, 5% CO2). Cell type-specific transfection (Thy1.1) was analyzed via flow cytometry below. Depicts the percentage of transfected cells (CD14+ monocytes, CD56+ NK cells, CD19+ B cells, CD3+ T cells, CD4+ T cells, or CD8+ T cells) within all transfected PBMCs (transfection, y-axis) for each formulation condition tested (see table below). [Figure 7]T cell targeting using LPLX with different lipids and linkers. LPLX was functionalized with aCD8 DARPin X NbAlfa using three different Alfa-tagged lipids and one control lipid without an Alfa tag: DSPE-PEG2k Alfa, DOPE-(G2SG2)2 Alfa, DOPE-(G2SG2)4 Alfa, and DOPE-(G2SG2)2. All LPLX contained Viromer L3 for RNA encapsulation (cargo: Thy1.1 / Luc RNA 1:1 w / w; RNA concentration: 0.1 μg / μL; N / P ratio: 15; ligand-to-cargo w / w ratio: 0, 1.5, or 2; ligand-to-functionalized lipid molar ratio: 1.5:1). Diameter and PDI were determined via DLS measurements. Successful RNA incorporation was verified via agarose gel electrophoresis. For transfection studies, 1e6 CD8+ Jurkat cells were seeded per well (96-well deep-bottom plate) in 100µl RPMI + 10% FBS. 3µl of each formulation (300ng RNA) was applied per well, followed by resuspension of the NP cell solution and incubation at 37°C for 10 minutes. 900µl of RPMI + 10% FBS was then added per well to reach a cell concentration of 1e6 cells / ml. 0.1e6 cells in 100µl were seeded on day 1 for luciferase expression readout. 0.3e6 cells in 300µl were seeded on day 1 for FACs-based detection of Thy1.1 expression. [Figure 8]T cell targeting using LNPs with different stealth lipids and alpha lipids. LNPs with different lipid compositions were formulated (cargo: Thy1.1 / Luc RNA 1:1 w / w; N / P ratio: 4; lipid mixture: HY501 / cholesterol / DSPC / stealth lipid / Alfa lipid [47.5 / 38 / 10 / 1.8 / 0.2], C16 PEG2k Ceramide or C14 Sar20 as stealth lipid and DSPE PEG2k Alfa, DOPE PSar20 Alfa, or DOPE PSar10 Alfa). The LNPs were equipped with aCD3 VHH x NbAlfa ligand via post-functionalization [w / w* = ligand-to-cargo ratio 0.48; ligand-to-functionalized lipid molar ratio 3.5:1]; RNA concentration: 0.1 μg / μl). All LNPs had diameters between 90 and 120 nm, with PDIs less than 0.3, as determined via DLS measurements. Successful RNA integration was verified via agarose gel electrophoresis. For transfection studies, 10 μl (1000 ng dose) of each formulation was prediluted in 50 μl X-Vivo 15 in an ultra-low attachment 96-well plate. 1 e6 thawed human PBMCs were diluted in 100% PHS and added to the nanoparticle dilutions. Plates were incubated overnight (37°C, 5% CO2). Cell type-specific transfection (Thy1.1) was analyzed via flow cytometry as follows: Percentage of transfected cells (CD14+ monocytes, CD56+ NK cells, CD19+ B cells, CD3+ T cells, CD4+ T cells, or CD8+ T cells) among all transfected PBMCs (transfection, y-axis) is depicted for each formulation condition tested (see table below). [Figure 9]T cell targeting using LNPs in a transgenic mouse model. LNPs with different lipid compositions and aCD3 VHH x NbAlfa ligands were formulated (cargo: Thy1.1 / Luc RNA 1:1 w / w; N / P ratio: 4; lipid mixture: DODMA / cholesterol / DOPE / C16 PEG2k-ceramide / DSPE-PEG2k-Alfa [40 / 48 / 10 / 1.8 / 0.2]). aCD3-VHH x NbAlfa was added via post-functionalization (w / w* = ligand-to-cargo ratio 1.15; ligand-to-functionalized lipid molar ratio 1.5:1); RNA concentration: 0.1 μg / μl). All LNPs had diameters between 90 and 120 nm, as determined via DLS measurements, with PDIs less than 0.3. Successful RNA incorporation was verified via agarose gel electrophoresis. Twenty micrograms of each formulation was injected intravenously into the tail vein of B6-hCD3EDG transgenic mice. Prior to injection, transgenic human CD3 expression on mouse T cells and specific binding of the anti-hCD3VHH x NbAlfa ligand to the transgenic receptor were verified in vitro on B6-hCD3EDG splenocytes. 18 h after LNP injection, animals were sacrificed, and splenocytes were analyzed for cell type-specific Thy1.1 expression via flow cytometry. Thy1.1 (transfection, y-axis) signal is depicted relative to CD69 activation marker expression on non-T / B cells, mCD19+ B cells, and mCD4+ or mCD8+ T cells. [Figure 10]B cell targeting using LNPs bearing algM. LNPs were formulated with or without ligands (cargo: Thy1.1 / Luc RNA 1:1 w / w; N / P ratio: 4; lipid mixture: DODMA / Chol / DOPE / C16-PEG-Ceramide / DSPE-PEG2k-Alfa [40 / 48 / 10 / 2-x / x = 0, 0.2]; aIgM-VHH X NbAlfa post-functionalization [w / w* = ligand-to-cargo ratio 0, 0.12, 0.25, 0.4]; final RNA concentration: 0.1 μg / μl). All formulation conditions lead to colloidally stable particles with diameters of 100–150 nm and PDIs below 0.5. Diameter and PDI were determined via DLS measurements. Successful RNA incorporation was verified via agarose gel electrophoresis. For transfection studies, 10 μl of each formulation was prediluted in 50 μl X-Vivo 15 in a 96-well deep-well plate. 1 e6 thawed human PBMCs were diluted in IMDM medium without FCS and added to the nanoparticle dilutions (1000 ng RNA). After 30 min of incubation (37°C, 5% CO2), 900 μl of IMDM + HI FCS + IL4 and IL21 was added per well, and cells were cultured for an additional 18 h (37°C, 5% CO2). Cell type-specific transfection (Thy1.1) was analyzed via flow cytometry below. Cell type-specific Thy1.1 (% transfection multiplied by MFI; y-axis divided by 100) signals are depicted in viable CD14+ monocytes, CD19+ B cells, and CD3+ T cells. [Figure 11] Freeze-thaw studies. Particle size analysis of functionalized lipoplexes subjected to two freeze-thaw cycles: (A) from −20°C to room temperature and (B) from −80°C to room temperature. [Figure 12]T cell targeting using LNPs with different stealth and Alfa lipids. LNPs with different lipid compositions were formulated (cargo: Thy1.1 RNA / Luc RNA / Np proxy Venus 1:1:2 w / w; N / P ratio: 6; lipid mixture: HY501 / cholesterol / DSPC / stealth lipid / Alfa lipid). The lipid ratio was selected as [47.5 / 40.5 / 10 / 1.8 / 0.2] for the following combinations of stealth and Alfa lipids: C16 PEG2k ceramide / DSPE PEG2k Alfa, DSPE PEG2k / DSPE PEG2k Alfa, DSPE pAEEA14 / DSPE pAEEA14 Alfa, or VitE pAEEA8 / DSPE pAEEA14 Alfa. The lipid ratios were selected as follows: [47.5 / 38.5 / 10 / 3.8 / 0.2] for the stealth lipid and Alfa lipid combinations: VitE PEG1k / DSPE PEG2k Alfa or VitE pAEEA8 / DSPE pAEEA14 Alfa. The LNPs were equipped with aCD3 VHH x NbAlfa ligand via post-functionalization [w / w* = ligand-to-cargo ratio 0.48]; RNA concentration: 0.1 μg / μl. All LNPs had diameters between 100 and 170 nm, with PDIs less than 0.4, as determined via DLS measurements. For transfection studies, 10 μl (1000 ng dose) of each formulation was prediluted in 50 μl X-Vivo 15 in an ultra-low attachment 96-well plate. 16 thawed human PBMCs were diluted in 50 μl of 100% coagulation PHS and added to the nanoparticle dilutions. After 30 min of incubation (37°C, 5% CO), 30 μl of each transfection reaction was transferred to a second ultra-low attachment 96-well plate, and 170 μl of X-Vivo 15 medium + 100 U / ml IL2 was added per well. Cell dilutions were cultured for an additional 18 h (37°C, 5% CO). Cell type-specific transfection (Thy1.1) was analyzed via flow cytometry below.The percentage of transfected cells (CD2 negative cells, CD19+ B cells, CD4+ T cells, and CD8+ T cells) within all transfected PBMCs (transfection, y-axis) is depicted for each formulation condition tested (see table below). [Figure 13A] (FIG. 13) Ligand-mediated transfection of T cells in vivo. Intramuscular injection of naked or LNP-formulated luciferase- and Thy1.1-encoding RNA mixtures (1:1 weight:weight mixture) in B6-hCD3EDG transgenic mice (1 μg RNA dose per injected side; 2 μg total RNA dose per mouse). Analysis was performed 18 h after injection. (A) Drainage of popliteal, inguinal, axillary, and brachial lymph nodes, as well as the spleen, analyzed by ex vivo bioluminescence imaging. [Figure 13B] (B) Cell type-specific transfection analyzed by flow cytometry via detection of delivered Thy1.1 RNA expression in immune cell subtypes within popliteal and inguinal lymph nodes, and the spleen. [Figure 13C] (C) T cell activation status analyzed by the mean fluorescence intensity of CD69 surface expression within the depicted organs (after staining with anti-CD69-APC antibody). LN, lymph node; LNP, lipid nanoparticles; NK, natural killer; PMN, polymorphonuclear cell. DETAILED DESCRIPTION OF THE INVENTION

[0078] Although the present disclosure will be further described in more detail below, it should be understood that the disclosure is not limited to the specific methods, protocols and reagents described herein, and these may vary.It should also be understood that the terms used herein are only intended to describe specific embodiments and are not intended to limit the scope of the present disclosure, which is limited only by the scope of the appended claims.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0079] The elements of the present disclosure are described in more detail below. While these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create further embodiments. The various described examples and preferred embodiments should not be construed as limiting the disclosure to only the explicitly described embodiments. The description should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered disclosed by the description of this application, unless the context dictates otherwise.

[0080] The practice of the present disclosure will employ, unless otherwise indicated, conventional chemical, biochemical, pharmacological, cell biology, immunological, and recombinant DNA techniques described in the art.

[0081] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprises" and variations such as "comprising" are understood to imply the inclusion of a stated feature, element, member, integer, or step or group of features, elements, members, integers, or steps, but not the exclusion of any other feature, element, member, integer, or step or group of features, elements, members, integers, or steps. The term "consisting essentially of" limits the scope of a claim or disclosure to the specified features, elements, members, integers, or steps and to those that do not materially affect the basic and novel characteristics of the claim or disclosure. The term "consisting of" limits the scope of a claim or disclosure to the specified features, elements, members, integers, or steps. The term "comprising" encompasses the term "consisting essentially of," which in turn encompasses the term "consisting of." Thus, in each occurrence in this application, the term "comprising" may be replaced with the term "consisting essentially of" or "consisting of." Similarly, in each occurrence in this application, the term "consisting essentially of" may be replaced with the term "consisting of."

[0082] As used in the context of describing this disclosure (particularly in the context of the claims), the terms "a," "an," "the," and similar references should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0083] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0084] The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better describe the disclosure and does not pose a limitation on the scope of the claimed disclosure. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0085] As used herein, the term "optional" or "optionally" means that the subsequently described event, circumstance, or condition may or may not occur, and that the description includes cases where said event, circumstance, or condition occurs and cases where it does not occur.

[0086] As used herein, "and / or" should be interpreted 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 interpreted as a specific disclosure of (i) X, (ii) Y, and (iii) each of X and Y, as if each were individually set forth herein.

[0087] In the context of the present disclosure, the term "about" indicates an interval 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 ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, e.g., ±0.01%. In some embodiments, "about" indicates a deviation of ±10% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±5% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±4% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±3% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±2% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±1% from the indicated numerical value. In some embodiments, "about" indicates a ±0.9% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.8% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.7% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.6% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.5% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.4% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.3% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.2% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.1% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.05% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.01% deviation from the indicated numerical value. As will be understood by one of ordinary skill in the art, such specific deviations from the numerical value of a given technical effect will depend on the nature of the technical effect. For example, natural or biological technical effects may generally have greater such deviations than artificial or engineered technical effects.

[0088] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and unless otherwise indicated herein, each separate value is incorporated herein as if it were individually listed herein.

[0089] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0090] The following provides definitions and embodiments that apply to all aspects of this disclosure. Terms defined below have the defined meanings unless otherwise indicated. Terms not defined have their art-wide accepted meanings.

[0091] As used herein, terms such as "reduce" or "inhibit" refer to the ability to cause an overall decrease in levels, for example, by about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, or about 75% or more. The term "inhibit" or similar phrases includes complete or essentially complete inhibition, i.e., a reduction to zero or essentially zero.

[0092] As used herein, terms such as "enhance" refer to the ability to cause an overall increase or enhancement in a level, for example, by at least about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 75% or more, or about 100% or more.

[0093] As used herein, "physiological pH" refers to a pH of 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.

[0094] As used in this disclosure, "% w / v" refers to weight-to-volume percent, a unit of concentration that measures the amount of solute in grams (g) expressed as a percentage of the total volume of a solution in milliliters (mL).

[0095] As used in this disclosure, "wt. %" refers to weight percent, a unit of concentration that measures the amount of a substance in grams (g) expressed as a percentage of the total weight of the entire composition in grams (g).

[0096] 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.

[0097] As used in this disclosure, "mol % total lipid" is defined as the ratio of the number of moles of one lipid component to the total number of moles of all lipids multiplied by 100. In this context, in some embodiments, the term "total lipid" includes lipids and lipid-like substances.

[0098] The term "ionic strength" refers to the mathematical relationship between the number of different ionic species in a particular solution and their respective charges. Thus, ionic strength, I, is calculated by the formula:

number

[0099] According to the present disclosure, the term "ionic strength" in some embodiments refers to the presence of monovalent ions. With respect to the presence of divalent ions, particularly divalent cations, their concentration or effective concentration (presence of free ions) due to the presence of chelating agents is, in some embodiments, sufficiently low to prevent degradation of nucleic acids. In some embodiments, the concentration or effective concentration of divalent ions is below the catalytic level for hydrolysis of phosphodiester bonds between nucleotides, such as RNA nucleotides. In some embodiments, the concentration of free divalent ions is 20 μM or less. In some embodiments, free divalent ions are absent or essentially absent.

[0100] "Osmolality" refers to the concentration of a particular solute expressed as osmoles of solute per kilogram of solvent.

[0101] The term "lyophilize" or "lyophilization" refers to the lyophilization of a substance by freezing the substance and then reducing the surrounding pressure (e.g., to less than 15 Pa, e.g., less than 10 Pa, less than 5 Pa, or 1 Pa or less) to cause the freezing medium in the substance to sublimate directly from the solid phase to the gas phase. Thus, the terms "lyophilize" and "freeze-dry" are used interchangeably herein.

[0102] The term "spray drying" refers to spray drying a substance by mixing a fluid and a (heated) gas that is atomized (atomized) in a vessel (spray dryer), and the solvent from the droplets formed evaporates, resulting in a dry powder.

[0103] The term "reconstitute" relates to the addition of a solvent, such as water, to a dried product to return it to a liquid state, such as its original liquid state.

[0104] The term "recombinant" in the context of this disclosure means "produced through genetic engineering." In some embodiments, "recombinant" in the context of this disclosure is not naturally occurring.

[0105] The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that exists in an organism (including viruses), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is naturally occurring. The term "found in nature" means "existing in nature", and includes known objects and objects that have not yet been discovered and / or isolated from nature, but may be discovered and / or isolated from natural sources in the future.

[0106] As used herein, the terms "room temperature" and "ambient temperature" are used interchangeably herein and refer to a temperature of at least about 15°C, e.g., from about 15°C to about 35°C, from about 15°C to about 30°C, from about 15°C to about 25°C, or from about 17°C to about 22°C. Such temperatures include 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, and 22°C.

[0107] The term "EDTA" refers to ethylenediaminetetraacetic acid disodium salt. All concentrations are given in terms of EDTA disodium salt.

[0108] The term "cryoprotectant" relates to a substance added to a formulation to protect the active ingredient during the freezing step.

[0109] The term "lyoprotectant" relates to a substance added to a formulation to protect the active ingredient during the drying step.

[0110] According to the present disclosure, the term "peptide" refers to a substance comprising about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids linked together by peptide bonds. The term "polypeptide" refers to large peptides, particularly peptides having at least about 151 amino acids. Both "peptides" and "polypeptides" are protein molecules. Therefore, the terms "peptide," "protein," and "polypeptide" are generally used synonymously herein.

[0111] The peptides and polypeptides disclosed herein can comprise linear or cyclized peptide sequences.

[0112] In some embodiments, the peptides disclosed herein comprise at least one cyclic portion, i.e., a polypeptide chain containing a cyclic bond sequence, referred to herein as a "cyclic peptide." The cyclic sequence can occur through a connection between the amino and carboxyl termini of the peptide; a connection between the amino terminus and a side chain; a connection between the carboxyl terminus and a side chain; or a connection between two side chains containing the sulfur groups of two cysteine ​​amino acids by forming a disulfide bond, or through more complex configurations.

[0113] In some embodiments, the peptides and polypeptides disclosed herein are composed of naturally occurring amino acids, non-naturally occurring amino acids, amino acid derivatives, and non-amino acid components, or mixtures thereof. In some embodiments, the peptides and polypeptides disclosed herein include amino acid mimetics and amino acid analogs. In some embodiments, the peptides and polypeptides disclosed herein include non-naturally occurring amino acid sequences that are resistant to enzymatic cleavage.

[0114] In some embodiments, one or more positions of a peptide or polypeptide disclosed herein are substituted with a non-naturally occurring amino acid. In some embodiments, the substituted amino acid is chemically related to the original residue (e.g., aliphatic, charged, basic, acidic, aromatic, hydrophilic) or is an isostere of the original residue.

[0115] In its broadest sense, as used herein, the term "amino acid" refers to a compound and / or substance that can be, is, or has been incorporated into a peptide, for example, through the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure HN-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid. In some embodiments, an amino acid is a D-amino acid. In some embodiments, an amino acid is an L-amino acid. A "standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides and polypeptides. A "non-standard amino acid" refers to any amino acid other than the standard amino acids, whether synthetically prepared or obtained from a natural source. In some embodiments, amino acids, including the carboxy- and / or amino-terminal amino acids in a peptide or polypeptide, may contain structural modifications compared to the general structures above. For example, in some embodiments, an amino acid may be modified relative to the general structure by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of an amino group, a carboxylic acid group, one or more protons, and / or a hydroxyl group). In some embodiments, such modifications may, for example, alter the circulating half-life of a peptide or polypeptide containing the modified amino acid compared to one containing an otherwise identical, unmodified amino acid. In some embodiments, such modifications do not significantly alter the relevant activity of a peptide or polypeptide containing the modified amino acid compared to one containing an otherwise identical, unmodified amino acid. As is clear from the context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid. In some embodiments, it may be used to refer to an amino acid residue of a peptide or polypeptide. The following table lists the 20 naturally occurring amino acids and their abbreviations: [Table 1] TIFF2025507295000004.tif18153

[0116] Generally, amino acids are L-amino acids, with D-amino acids being designated by the prefix "D." The prefix "homo" or "h" designates α-amino acids that are otherwise similar to common α-amino acids but contain one additional methylene group in the carbon chain.

[0117] As used herein, "Orn" means ornithine or 2,5-diaminopentanoic acid, "Dab" means 2,4-diaminobutanoic acid, "Dap" means 2,3-diaminopropanoic acid, "hLys" means 2,7-diaminoheptanoic acid, "hCys" means 2-amino-4-mercaptobutanoic acid, and "Pen" means penicillamine or 2-amino-3-methyl-3-sulfanylbutanoic acid.

[0118] It may also be possible to include non-peptide bonds and other chemical modifications.For example, part or all of a peptide or polypeptide can be synthesized as a peptide mimetic, such as a peptoid (see, for example, Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89:9367-71 and Horwell (1995) Trends Biotechnol. 13:132-4).A peptide or polypeptide can include one or more (for example, all) non-hydrolyzable bonds.Many non-hydrolyzable peptide bonds are known in the art, along with procedures for synthesizing peptides containing such bonds. Exemplary non-hydrolyzable bonds include -[CHNH]-reduced amide peptide bond, -[COCH]-ketomethylene peptide bond, -[CH(CN)NH]-(cyanomethylene)amino peptide bond, -[CHCH(OH)]-hydroxyethylene peptide bond, -[CHO]-oxymethylene peptide bond, and -[CHS]-thiomethylene peptide bond (see, e.g., U.S. Pat. No. 6,172,043).

[0119] As used herein, the term "amide" refers to a group of the formula "-NHC(O)-".

[0120] The term "thioamide" refers to a group of the formula "-NHC(S)-".

[0121] As used herein, the terms "disulfide bond," "disulfide bridge," or "disulfide" include a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge with a second thiol group.

[0122] The term "ether" refers to a group or compound that has an oxygen between two carbon atoms.

[0123] The term "thioether" refers to a group or compound that has a sulfur between two carbon atoms.

[0124] The term "ester" refers to a compound derived from a carboxylic acid and an alcohol by linking the hydroxyl group of the -C(=O)OH group in the former with the hydroxy group of the latter, with formal loss of water. The term therefore refers to the group -C(O)O-.

[0125] The term "thioester" refers to the group -C(O)S-.

[0126] The term "triazole" refers to a chemical compound that incorporates any heterocyclic structure having a five-membered ring of two carbon atoms and three nitrogen atoms (eg, 1,2,3-triazole).

[0127] The term "portion" refers to a fraction. With respect to a particular structure such as an amino acid sequence or a protein, the term "portion" may refer to a contiguous or discontinuous fraction of said structure.

[0128] The terms "portion" and "fragment" are used interchangeably herein and refer to a continuous element. For example, a portion of a structure, such as an amino acid sequence or protein, refers to a continuous element of said structure. When used in reference to a composition, the term "portion" refers to a portion of the composition. For example, a portion of a composition can be any portion between 0.1% and 99.9% of the composition (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%).

[0129] With respect to an amino acid sequence (peptide or polypeptide), the term "fragment" refers to a portion of the amino acid sequence, i.e., a sequence representing an amino acid sequence truncated at the N-terminus and / or C-terminus. A C-terminally truncated fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. An N-terminally truncated fragment (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains an initiation codon that serves to initiate translation. A fragment of an amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues from the amino acid sequence. A fragment of an amino acid sequence comprises, for example, at least 6, particularly at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from the amino acid sequence. Fragments of an amino acid sequence include, for example, sequences of up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55 consecutive amino acids of the amino acid sequence.

[0130] As used herein, "variant" with respect to an amino acid sequence (peptide or polypeptide) means an amino acid sequence that differs from a parent amino acid sequence by at least one amino acid (e.g., a different amino acid, or a modification of the same amino acid). The parent amino acid sequence can be a native or wild-type (WT) amino acid sequence, or can be a modified version of the wild-type amino acid sequence. In some embodiments, the variant amino acid sequence has at least one amino acid difference compared to the parent amino acid sequence, e.g., 1 to about 20 amino acid differences compared to the parent, e.g., 1 to about 10 or 1 to about 5 amino acid differences.

[0131] By "wild-type" or "WT" or "native" as used herein with respect to an amino acid sequence (peptide or polypeptide) is meant an amino acid sequence found in nature, including allelic variations. A wild-type amino acid sequence, peptide, or polypeptide has an amino acid sequence that has not been intentionally modified.

[0132] For purposes of this disclosure, a "variant" of an amino acid sequence (peptide or polypeptide) can include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translationally modified variants, conformational variants, isoform variants, allelic variants, species variants, and species homologs, particularly those that occur naturally. The term "variant" particularly includes fragments of an amino acid sequence.

[0133] Amino acid insertion variants include the insertion of a single or two or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted at a specific site in the amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible. Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, for example, 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, for example, 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion can be at any position in the protein. Amino acid deletion variants containing deletions at the N- and / or C-termini of a protein are also called N- and / or C-terminal truncation variants. Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Modifications at positions within the amino acid sequence that are not conserved between homologous peptides or peptides and / or substitutions of amino acids with other amino acids with similar properties are preferred. In some embodiments, amino acid changes in peptide and polypeptide variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve substitutions of one member of a family of amino acids whose side chains are related. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. In some embodiments, conservative amino acid substitutions include substitutions within the following groups: Glycine, Alanine; valine, isoleucine, leucine; Aspartic acid, glutamic acid; Asparagine, glutamine; Serine, threonine; lysine, arginine; and Phenylalanine, tyrosine.

[0134] In some embodiments, the degree of similarity, such as identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence is at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the degree of similarity or identity is given over an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is given for, for example, at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments, consecutive amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, such as sequence identity, can be performed using tools known in the art, for example, using best sequence alignment, for example, Align, using standard settings, preferably EMBOSS::Needle, matrix:Blosum62, gap open 10.0, gap extension 0.5.

[0135] "Sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences.

[0136] The terms "% identical" and "% identity" or similar terms are intended to refer to the percentage of nucleotides or amino acids that are identical in the optimal alignment between the sequences being compared.The percentage is purely statistical, and the differences between two sequences may be, but are not necessarily, randomly distributed over the entire length of the sequences being compared.Comparing two sequences is usually carried out by comparing sequences over a segment or "comparison window" after optimal alignment to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually or with the aid of an algorithm, such as the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 88, 2444, or a computer program using the above algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithms available at the United States National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq).In some embodiments, the algorithm parameters used for the BLASTN algorithm on the NCBI website include: (i) an expectation threshold set to 10; (ii) a word size set to 28; (iii) a maximum match within the query range set to 0; (iv) match / mismatch scores set to 1, -2; (v) a gap cost set to linear; and (vi) a filter for low-complexity regions being used. In some embodiments, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) an expectation threshold set to 10; (ii) a word size set to 3; (iii) a maximum match within the query range set to 0; (iv) a matrix set to BLOSUM62; (v) gap costs set to presence: 11, extension: 1; and (vi) a conditional composition score matrix adjustment.

[0137] The percent identity is obtained by determining the number of identical positions where the compared sequences match, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100.

[0138] In some embodiments, the degree of similarity or identity is given for a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments, consecutive nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.

[0139] Homologous amino acid sequences, according to the present disclosure, exhibit an identity of at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, for example at least 95%, at least 98 or at least 99% of the amino acid residues.

[0140] The amino acid sequence variants described herein can be readily prepared by those skilled in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to prepare peptides or polypeptides with substitutions, additions, insertions, or deletions is described in detail, for example, in Molecular Cloning: A Laboratory Manual, 4th Edition, M.R. Green and J. Sambrook et al. (1989), eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012. Furthermore, the peptides, polypeptides, and amino acid variants described herein can be readily prepared using known peptide synthesis techniques, for example, by solid-phase synthesis and similar methods.

[0141] In some embodiments, a fragment or variant of an amino acid sequence (peptide or polypeptide) is a "functional fragment" or "functional variant." The term "functional fragment" or "functional variant" of an amino acid sequence refers to any fragment or variant that exhibits one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., is functionally equivalent. With respect to sequences of binding agents such as antibodies, one particular function is one or more binding activities exhibited by the amino acid sequence from which the fragment or variant is derived. As used herein, the term "functional fragment" or "functional variant" specifically refers to a variant molecule or sequence that contains an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of a parent molecule or sequence and still performs one or more functions of the parent molecule or sequence, e.g., is capable of binding to a target molecule. In some embodiments, alterations to the amino acid sequence of the parent molecule or sequence do not significantly affect or change the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present, for example, the function of the functional fragment or functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence, however, in other embodiments, the function of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0142] An amino acid sequence (peptide or polypeptide) "derived from" a specified amino acid sequence (peptide or polypeptide) refers to the origin of the initial amino acid sequence. In some embodiments, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to the particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence or a fragment thereof. For example, it will be understood by those skilled in the art that sequences suitable for use herein may be modified to differ in sequence from the naturally occurring or native sequence from which they are derived while retaining the desired activity of the native sequence.

[0143] In some embodiments, "isolated" means removed (e.g., purified) from a natural state or from an artificial composition, such as a composition from a manufacturing process. For example, a nucleic acid, peptide, or polypeptide that is naturally present in a living animal is not "isolated," but the same nucleic acid, peptide, or polypeptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid, peptide, or polypeptide can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0144] The term "bind" or "binding" refers to a non-covalent interaction with a target. In some embodiments, the term "bind" or "binding" refers to specific binding. As used herein, the term "specific binding" or "specifically binds" refers to a molecule, such as an antibody or antigen receptor, that recognizes a specific target molecule but does not substantially recognize or bind other molecules in a sample or subject. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. However, such species cross-reactivity does not in itself change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not in itself change the classification of the antibody as specific.

[0145] In some cases, the terms "specific binding" or "specifically binds" can be used in reference to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) in the chemical species; for example, antibodies recognize and bind to specific protein structures rather than proteins in general. If an antibody is specific for epitope "A," then in a reaction involving labeled "A" and the antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A that binds to the antibody.

[0146] As used herein, the terms "bind" or "capable of binding" typically refer to binding of about 10, as determined using biolayer interferometry (BLI) or, for example, as determined using surface plasmon resonance (SPR) technology on a BIAcore 3000 instrument. -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 M or less, about 10 -10 M or less, or about 10 -11 M or even lower K D In some embodiments, the binding moiety or agent binds with an affinity corresponding to a K that is at least 10-fold lower, e.g., at least 100-fold lower, e.g., at least 1,000-fold lower, e.g., at least 10,000-fold lower, e.g., at least 100,000-fold lower, e.g., at least 100,000-fold lower, than its affinity for binding to a non-specific target (e.g., BSA, casein). D The target binds to a given target with an affinity corresponding to

[0147] As used herein, "k" d ”(seconds -1 The term k ) refers to the dissociation rate constant of a particular interaction, e.g., an antibody-antigen interaction. off Also called value.

[0148] As used herein, "K" D The term "" (M) refers to the dissociation rate constant of a particular interaction, for example, an antibody-antigen interaction.

[0149] In general, the terms "bind" or "binding" and "target" or "targeting" are used interchangeably herein.

[0150] The term "genetic modification" or simply "modification" includes the transfection of cells with nucleic acids. The term "transfection" refers to the introduction of nucleic acids, e.g., DNA and / or RNA, into cells. For purposes of this disclosure, the term "transfection" also includes the introduction of nucleic acids into cells or the uptake of nucleic acids by such cells, and the cells may be present in a subject, e.g., a patient, or the cells may be present in vitro, e.g., outside the patient. Thus, according to this disclosure, cells for transfection of nucleic acids described herein can be present in vitro or in vivo, e.g., the cells can form part of a patient's organ, tissue, and / or body. According to this disclosure, transfection can be transient or stable. In some applications of transfection, it is sufficient for the transfected genetic material to be expressed only transiently. RNA can be transfected into cells to transiently express its encoded protein. Nucleic acids introduced during the transfection process are typically not integrated into the nuclear genome, so the foreign nucleic acid is diluted or degraded by mitosis. Cells that allow episomal amplification of nucleic acids significantly reduce the dilution rate. If it is desired that transfected nucleic acid actually remains in the genome of the cell and its daughter cells, stable transfection must occur.Such stable transfection can be achieved, for example, by using a virus-based system or a transposon-based system for transfection.Generally, the cells that are genetically modified to express antigen receptors are stably transfected with the nucleic acid encoding the antigen receptor.RNA can be transfected into cells to transiently express the encoded protein.

[0151] As used herein, the terms "linked," "fused," or "fusion" are used interchangeably and refer to the joining of two or more elements or components or domains.

[0152] The term "fusion protein" as used herein refers to a polypeptide or protein comprising two or more subunits. Preferably, the fusion protein is a translational fusion between two or more subunits. A translational fusion can be generated by genetically engineering the coding nucleotide sequence of one subunit in the same reading frame with the coding nucleotide sequence of another subunit. The subunits can be interspersed with a linker.

[0153] As used herein, "endogenous" refers to any substance that is produced from or within an organism, cell, tissue, or system.

[0154] As used herein, the term "exogenous" refers to any substance that is introduced into or produced outside of an organism, cell, tissue, or system.

[0155] The term "autologous" is used to refer to something derived from the same subject. For example, "autologous transplantation" refers to the transplantation of tissue or organs derived from the same subject. Such procedures are advantageous because they overcome immunological barriers that would otherwise result in rejection.

[0156] The term "allogeneic" is used to describe something that is derived from different individuals of the same species. Two or more individuals are said to be allogeneic to one another if the genes at one or more loci are not identical.

[0157] The term "syngeneic" is used to describe individuals or tissues that have the same genotype, i.e., derived from identical twins or the same inbred strain of animals, or tissues thereof.

[0158] The term "xenogeneic" is used to describe something that is made up of multiple dissimilar elements. As an example, transferring bone marrow from one individual to another constitutes a xenogeneic transplant. A xenogeneic gene is a gene that originates from a source other than the subject.

[0159] According to various embodiments of the present disclosure, a nucleic acid encoding a peptide or polypeptide is taken up or introduced, i.e., transfected or transduced, into a cell, which may be present in vitro or in a subject, resulting in expression of the peptide or polypeptide. The cell may, for example, express the encoded peptide or polypeptide intracellularly (e.g., in the cytoplasm and / or nucleus), secrete the encoded peptide or polypeptide, and / or express it on its surface. In some embodiments, if the encoded peptide or polypeptide is an antigen receptor, the cell expresses the antigen receptor on the cell surface.

[0160] In accordance with the present disclosure, terms such as "expressing nucleic acid" and "encoding nucleic acid" or similar terms are used interchangeably herein and mean that, with respect to a particular peptide or polypeptide, the nucleic acid is capable of being expressed to produce said peptide or polypeptide when present in an appropriate environment, e.g., a cell.

[0161] As used herein, the term "expression" includes the transcription and / or translation of a particular nucleotide sequence.

[0162] In the context of the present disclosure, the term "transcription" relates to the process by which the genetic code in a DNA sequence is transcribed into RNA (particularly mRNA), which can then be translated into peptides or polypeptides.

[0163] With respect to RNA, the terms "expression" or "translation" refer to the process in a cell's ribosomes by which a chain of mRNA directs the assembly of a sequence of amino acids to make a peptide or polypeptide.

[0164] The medical preparations described herein, particularly kits, may include instruction materials or instructions. As used herein, "instruction materials" or "instructions" include publications, records, drawings, or any other medium of expression that can be used to communicate the usefulness of the compositions and methods of the present disclosure. The instruction materials of the kits of the present disclosure may be, for example, attached to a container containing the composition / formulation of the present disclosure, or may be shipped together with a container containing the composition / formulation. Alternatively, the instruction materials may be shipped separately from the container, with the intention that the instruction materials and the composition will be used in conjunction with each other by the recipient.

[0165] The term "mean diameter" refers to the average hydrodynamic diameter of particles measured by dynamic light scattering (DLS) with data analysis using the so-called cumulant algorithm, which has a length dimension, the so-called Z 平均 , and provides a dimensionless polydispersity index (PDI) as a result (Koppel, D., J. Chem. Phys. 57, 1972, pp. 4814-4820, ISO 13321). Here, the "average diameter", "diameter" or "size" of a particle is this Z 平均 Used synonymously with the value of

[0166] In some embodiments, the "polydispersity index," as mentioned in the definition of "mean diameter," is calculated based on dynamic light scattering measurements by so-called cumulant analysis. Under certain prerequisites, it can be considered as a measure of the size distribution of an ensemble of nanoparticles.

[0167] The "radius of gyration" of the particle around the axis of rotation (R g ) is the radial distance from the axis of rotation of the point at which the moment of inertia of a particle about a given axis is the same as its actual mass distribution, if the entire mass of the particle were assumed to be concentrated. Mathematically, R g is the root mean square distance of a particle's components from either its center of mass or a given axis. For example, if the particle is at a fixed distance s from the center of mass, i Mass m located at iFor a polymer consisting of n mass elements (i=1, 2, 3, ..., n), R g is the s over all mass elements i 2 is the mass-averaged square root of and can be calculated as follows:

number

[0168] The radius of gyration can be determined experimentally or calculated, for example, by using light scattering. In particular, for small scattering vectors

number

number

[0169] The "hydrodynamic radius" (sometimes called the "Stokes radius" or "Stokes-Einstein radius") of a particle is the radius of a hypothetical hard sphere diffusing at the same rate as the particle. The hydrodynamic radius is related to the particle's mobility, taking into account not only size but also solvent effects. For example, a smaller charged particle with stronger hydration may have a larger hydrodynamic radius than a larger charged particle with weaker hydration. This is because the smaller particle drags more water molecules with it as it moves through the solution. Since the actual dimensions of a particle in a solvent cannot be measured directly, the hydrodynamic radius is determined by the Stokes-Einstein equation:

number

[0170] As used herein, the expression "light scattering" refers to a physical process in which light is forced to deviate from a straight line trajectory by one or more paths due to local inhomogeneities in the medium through which it passes.

[0171] The term "UV" means ultraviolet and refers to the band of the electromagnetic spectrum having wavelengths between 10 nm and 400 nm, i.e., shorter than those of visible light but longer than X-rays.

[0172] The expression "multi-angle light scattering" or "MALS" as used herein relates to a technique for measuring light scattered at multiple angles by a sample. "Multi-angle" in this context means that the scattered light can be detected at different discrete angles, as measured, for example, by a single detector moving over a range that includes a selected specific angle, or by an array of detectors fixed at specific angular positions. In certain embodiments, the light source used in MALS is a laser source (MALLS: Multi-Angle Laser Light Scattering). Based on the MALS signal of a composition containing particles, the radius of gyration (R) can be calculated by using an appropriate format (e.g., Zimm plot, Berry plot, or Debye plot). g ), and thus it is possible to determine the size of the particles. Preferably, the Zimm plot is calculated using the following formula:

number

number

number

[0173] As used herein, the term "dynamic light scattering" or "DLS" refers to a technique for determining particle size and size distribution profiles, particularly with respect to the hydrodynamic radius of particles. A monochromatic light source, usually a laser, is incident on a sample through a polarizer. The scattered light then passes through a second polarizer, where it is detected, and the resulting image is projected onto a screen. Particles in solution strike the light and diffract it in all directions. The diffracted light from the particles can interfere constructively (bright areas) or destructively (dark areas). This process is repeated over short time intervals, and the resulting set of speckle patterns is analyzed by an autocorrelator, which compares the light intensity at each spot over time.

[0174] As used herein, the term "static light scattering" or "SLS" refers to a technique for determining particle size and size distribution profiles, particularly with respect to the particle's radius of gyration and / or molar mass. A high-intensity monochromatic light, usually a laser, is emitted into a solution containing the particles. One or more detectors are used to measure the scattered intensity at one or more angles. The angular dependence is necessary to obtain accurate measurements of both the molar mass and size of all macromolecules within a radius. Therefore, simultaneous measurements at several angles relative to the direction of incident light, known as multi-angle light scattering (MALS) or multi-angle laser light scattering (MALLS), are generally considered the standard implementation of static light scattering.

[0175] targeting compound The particles described herein containing a nucleic acid payload to be delivered comprise a hydrophobic moiety (e.g., a lipid) to which a binding moiety is covalently attached. This hydrophobic moiety to which a binding moiety is covalently attached is also referred to herein as a "targeting compound." The hydrophobic moiety of the targeting compound refers to the portion of the targeting compound that is incorporated into the particle containing the nucleic acid payload. The binding moiety of the targeting compound refers to the portion of the targeting compound that binds to the target cell or forms a binding partner for a docking compound that binds to the target cell. Generally, the targeting compound is incorporated non-covalently into the particle containing the payload, i.e., it forms an integral part of the particle, and the binding moiety of the targeting compound is covalently attached to the hydrophobic moiety in such a manner that it is available to bind to the target cell or the docking compound.

[0176] In some embodiments, the binding moiety of the targeting compound comprises a peptide or protein (eg, an antibody or antibody fragment or a peptide tag).

[0177] In some embodiments, the binding portion of the targeting compound comprises a peptide or protein (e.g., an antibody or antibody fragment or a peptide tag) and is chemically linked, e.g., via a linker, to a hydrophobic moiety (e.g., a lipid).

[0178] The targeting compounds described herein comprise a hydrophobic moiety (e.g., a lipid moiety) that allows them to be anchored in particles. In some embodiments, the hydrophobic moiety comprises a moiety selected from vitamin E, dialkylamines such as dimyristylamine (DMA), diacylglycerides such as 1,2-dimyristoyl-sn-glycerol (DMG), and 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.

[0179] In some embodiments, the targeting compounds described herein have a phospholipid, such as a biodegradable phospholipid, such as phosphatidylethanolamine, as the hydrophobic group (e.g., lipid). In some embodiments, the targeting compounds described herein have a glycerophospholipid as the hydrophobic group (e.g., lipid). In some embodiments, the phospholipid is selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine), and mixtures thereof. In some embodiments, DSPE is used as the phospholipid due to its stability qualities in the particles described herein. Furthermore, compounds having at least one alkyl chain that provides hydrophobic anchoring to the particles described herein can be used as the hydrophobic group (e.g., lipid).

[0180] In some embodiments, the targeting compound comprises a polymer. In some embodiments, the hydrophobic portion of the targeting compound (e.g., a lipid) and the binding portion of the targeting compound are connected via a polymer.

[0181] In some embodiments, the polymer is a hydrophilic polymer, and the targeting 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 be anchored into the particle, and a hydrophilic component of the polymer facing the outside of the particle, which confers hydrophilic properties to the particle's surface. In some embodiments, the amphiphilic derivative of the polymer is inserted into the particle via its hydrophobic end. As a result, the polymer component faces the outside of the particle and forms a protective hydrophilic shell surrounding the particle. In some embodiments, the polymer portion of the amphiphilic derivative contributes to the particle's stealth properties. In some embodiments, the particles described herein have a plasma half-life of greater than 2 hours, e.g., 3 to 10 hours. This characteristic advantageously allows the particles to accumulate in target cells and release their contents (payload) into the target cells within a reasonable time. The effectiveness of the targeted delivery methods described herein is therefore increased as a result.

[0182] The term "stealth" is used herein to describe the ability of the particles described herein to not be detected and then sequestered and / or degraded, or to be poorly detected and then sequestered and / or degraded, and / or to be slowly detected and then sequestered and / or degraded by the immune system of a host to which the particles are administered.

[0183] Macrophages constitute one of the most important components of the immune system and play a major role in the clearance of foreign particles, including liposomes and other colloidal particles, from the circulation. At the molecular level, particle clearance occurs in two steps: opsonization by the deposition of serum proteins (or "opsonins") on the particle's surface, followed by recognition and capture of the opsonized particle by macrophages.

[0184] Modification of the particle surface with hydrophilic and flexible polymer chains, such as poly(ethylene glycol) type polymers, confers steric protection to the particle by preventing opsonins from reaching the particle surface.

[0185] In some embodiments, the amphiphilic derivatives of the polymers used herein have a hydrophobic group (e.g., lipid) as specified herein. In some embodiments, the amphiphilic derivatives of the polymers used herein have a phospholipid, such as a biodegradable phospholipid, such as phosphatidylethanolamine, as the hydrophobic group (e.g., lipid). In some embodiments, the phospholipid is selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine), and mixtures thereof. In some embodiments, DSPE is used as the phospholipid for its stability qualities in the particles described herein. Furthermore, compounds having at least one alkyl chain that provides hydrophobic anchoring to the particles described herein can be used as the hydrophobic group (e.g., lipid).

[0186] In some embodiments, polymers for use herein are 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.

[0187] In some embodiments, the polymer is designed to sterically stabilize the particle by forming a protective hydrophilic layer, hi some embodiments, the polymer can reduce particle association with serum proteins and / or resulting uptake by the reticuloendothelial system when such particles are administered in vivo.

[0188] In some embodiments, PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In some embodiments, PEG is unsubstituted. In some embodiments, PEG is substituted, for example, with one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In some embodiments, 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 6000, in another embodiment about 150 to about 5000, in another embodiment about 150 to about 4000, in another embodiment about 150 to about 3000, in another embodiment about 300 to about 3000, in another embodiment about 1000 to about 3000, and in yet another embodiment about 1500 to about 2500.

[0189] In some embodiments, the PEG portion of the amphiphilic derivative of the polymer has a molecular weight of 1000 or greater. In some embodiments, the PEG portion of the amphiphilic derivative of the polymer has 10 or more units of the formula (O-CH-CH) n In some embodiments, the PEG comprises 20 to 200 ethylene oxide units, for example, about 45 ethylene oxide units.

[0190] In some embodiments, the PEG comprises "PEG2k," also referred to as "PEG2000," having an average molecular weight of about 2000 daltons.

[0191] In some embodiments, DSPE-PEG 2000 , DSPE-PEG 3000 and DSPE-PEG 5000 is used as an amphiphilic derivative of the polymer.

[0192] In some embodiments, pSar comprises between 2 and 200 sarcosine units, such as between 5 and 100 sarcosine units, between 10 and 50 sarcosine units, between 15 and 40 sarcosine units, such as about 23 sarcosine units.

[0193] In some embodiments, pSar has the following general formula: [ka] where s is the number of sarcosine units. Includes the structure of

[0194] In some embodiments, the POX and / or POZ polymers may be selected from the group consisting of 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 1 and 30, 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.

[0195] In some embodiments, the POX and / or POZ polymers have the following general formula: [ka] (wherein a is an integer of 1 to 2; R 11 is alkyl, especially C 1-3 alkyl, e.g., methyl, ethyl, iso-propyl, or n-propyl, independently selected for each repeat unit; m refers to the number of POX and / or POZ repeat units. Includes.

[0196] In some embodiments, the POX and / or POZ polymer is a polymer of POX and includes repeat units of the general formula: [ka]

[0197] In some embodiments, the POX and / or POZ polymer is a polymer of POZ and includes repeat units of the general formula: [ka]

[0198] In any of the above embodiments of the formula, m (i.e., the number of repeat units in the polymer) is preferably 2 to 190, for example, 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 16 In certain embodiments, m is 0, 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. In certain embodiments, m is 2 to 180, e.g., 4 to 160, 6 to 140, 8 to 120, or 10 to 100, e.g., 20 to 80, 30 to 70, or 40 to 50.

[0199] In some embodiments, the POX and / or POZ polymers have the following general formula: [ka] (wherein the formula, the number of repeating units shown on the left in the copolymer is 1 to 199; the number of repeating units of the formula on the right in the copolymer is 1 to 199; and the sum of the number of repeating units of the formula on the left and the number of repeating units of the formula on the right in the copolymer is 2 to 200). It is a copolymer containing repeating units of the formula:

[0200] In some embodiments of the oxazolinylated and / or oxazinylated hydrophobic moiety (e.g., lipid), the number of repeat units of the left formula in the copolymer is 1 to 179, for example, 1 to 159, 1 to 139, 1 to 119, or 1 to 99; the number of repeat units of the right formula in the copolymer is 1 to 179, for example, 1 to 159, 1 to 139, 1 to 119, or 1 to 99; and the sum of the number of repeat units of the left formula and the number of repeat units of the right formula in the copolymer is 2 to 180, for example, 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.

[0201] In some of the above embodiments, R in each occurrence (i.e., each repeat unit) 11 can be the same alkyl group (e.g., R 11 In some alternative embodiments, R in at least one repeat unit is methyl. 11 is the R in another repeat unit 11 (e.g., R 11 is one specific alkyl (e.g., ethyl), and for at least one different repeat unit, R 11 are different specific alkyls (e.g., methyl). For example, each R 11 may be selected from two different alkyl groups (e.g., methyl and ethyl), and all R 11 are not the same alkyl.

[0202] In any of the above embodiments, R 11 is preferably methyl or ethyl, more preferably methyl. Thus, in some embodiments, each R 11 is methyl, or each R 11 is ethyl. In some alternative embodiments, R 11is independently selected from methyl and ethyl for each repeat unit, and R 11 is methyl, and in at least one repeat unit, R 11 is ethyl.

[0203] 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 a derivative thereof.

[0204] In some embodiments, the polymer has the following general formula: [ka] wherein X2 and X1 together are an optionally substituted amide, an optionally substituted thioamide, or an ester; Y is -CH2-, -(CH2)2-, or -(CH2)3-; z is 2 to 24; and (n is 1 to 100) Includes.

[0205] In some embodiments, (i)X 1 If is -C(O)-, then X 2 Ha-NR 1 - and; (ii) X 1 Ga-NR 1 -If X 2 is -C(O)-; (iii)X 1 If is -C(S)-, then X 2 Ha-NR 1 - and; (iv) X 1 Ga-NR 1 -If X 2 is -C(S)-; (v)X 1 If is -C(O)-, then X 2 is -O-; or (vi)X 1 If is -O-, then X 2 is -C(O)-; R 1 is hydrogen or C 1-8 It is alkyl.

[0206] In some embodiments, X 1 is -C(O)- and X 2 Ha-NR 1 - and R 1 is hydrogen or C 1-8 In some embodiments, X is alkyl. 1 is -C(O)- and X 2 Ha-NR 1 - and R 1 is hydrogen or methyl. In some embodiments, X 1 is -C(O)- and X 2 Ha-NR 1 - and R 1 is hydrogen.

[0207] In some embodiments, Y is —CH 2 — or —(CH 2 ) 2 —. In some embodiments, Y is —CH 2 —.

[0208] In some embodiments, the polymer has the following general formula: [ka] (In the formula, R 1 is hydrogen or C 1-8 is alkyl; z is 2 to 24; and n is 1 to 100) Includes.

[0209] 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.

[0210] In some embodiments, the polymer has the following general formula: [ka] (In the formula, R 1 is hydrogen or C 1-8 is alkyl; and n is 1 to 100) Includes.

[0211] In some embodiments of the above formula, R 1 is hydrogen or methyl. In some embodiments, R 1 is hydrogen.

[0212] In some embodiments, the polymer has the following general formula: [ka] (In the formula, n is 1 to 100) Includes.

[0213] 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.

[0214] In some embodiments, the molar fraction of the amphiphilic derivative of the polymer incorporated into the particle is 0.5 to 20 mol %, preferably 1 to 10 mol %, of the lipid molecules constituting the particle.

[0215] In some embodiments, the targeting compound has the following general formula: L-X1-P-X2-B (In the formula, P comprises a polymer; L comprises a hydrophobic moiety (e.g., a lipid) attached to a first end of the polymer; B comprises a linking moiety attached to the second end of the polymer; X1 is absent or a first linking moiety; and X2 is absent or a second linking moiety Includes.

[0216] In some embodiments, X1 comprises a carbonyl group. In some embodiments, L comprises a phosphatidylethanolamine, which can be linked to P by an amide group.

[0217] In some embodiments, X2 comprises the reaction product of a thiol or cysteine ​​reactive group, eg, a maleimide group, with a thiol or cysteine ​​group of the compound comprising the binding moiety.

[0218] In some embodiments, L comprises a lipid as described above. In some embodiments, L comprises DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine), which can be linked to P by an amide group.

[0219] In some embodiments, P comprises a polymer as described above. In some embodiments, P comprises a polymer that provides stealth properties, extends circulating half-life, and / or reduces nonspecific protein binding or cell adhesion. In some embodiments, 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, P comprises polyethylene glycol (PEG); e.g., PEG as described above.

[0220] In some embodiments, L-X1-P comprises an amphiphilic derivative of the polymer described above. In some embodiments, the amphiphilic derivative of the polymer comprises a conjugate of disteroyl-glycero-phosphoethanolamine (DSPE) and a polymer, such as a polymer described above. In some embodiments, the amphiphilic derivative of the polymer comprises a disteroyl-glycero-phosphoethanolamine-polyethylene glycol conjugate (DSPE-PEG).

[0221] In some embodiments, the targeting compound can be obtained by reacting a thiol or cysteine ​​group of a compound comprising a binding moiety with 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).

[0222] In some embodiments, the thiol or cysteine ​​reactive group comprises a maleimide group.

[0223] In some embodiments, the PEG reagent comprises DSPE-PEG-maleimide. In some embodiments, the compound comprising the binding moiety has the formula SH(CH) n In some embodiments, n is 2.

[0224] In some embodiments, the targeting compound comprises 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)] and a compound of formula SH(CH) n and a compound comprising C(O)—B, where n is in the range of 1 to 5, and B comprises a linking moiety. In some embodiments, n is 2.

[0225] In some embodiments, the targeting compound (the hydrophobic moiety to which the binding moiety is covalently attached) has the following general formula: L-X1-P-X2-B wherein L, X1, P, and B are as defined above, and X2 comprises a thiosuccinimide moiety. Includes.

[0226] In some embodiments, the targeting compound (the hydrophobic moiety to which the binding moiety is covalently attached) has the following general formula: [ka] wherein B includes a bond moiety. Includes.

[0227] In some embodiments of the above formula, B comprises a moiety comprising the structure -N-peptide-C(O)-NH2.

[0228] In some embodiments, the targeting compound (the hydrophobic moiety to which the binding moiety is covalently attached) has the following general formula: [ka] wherein P, X2, and B are as defined above, and R1 and R2 independently comprise alkyl moieties. In some embodiments, at least one, e.g., each alkyl moiety is linear or branched, preferably linear. In some embodiments, at least one, e.g., each alkyl moiety has at least 8 carbon atoms, e.g., 8 to 24, e.g., 10 to 18 carbon atoms. Preferably, at least one, e.g., each alkyl moiety is an alkyl moiety of a fatty acid alcohol, more preferably, at least one, e.g., each alkyl moiety is an alkyl moiety of a fatty acid alcohol having at least 8 carbon atoms, e.g., 8 to 24, e.g., 10 to 18 carbon atoms. An example of an alkyl moiety is -(CH2) 17 CH3 (Stearyl), -(CH2) 15 CH3 (palmityl), and -(CH2) 13 In some embodiments, R1R2N- in the above formula is 1,2-dimyristylamine, where both alkyl groups are -(CH2).13 CH3 (myristyl).

[0229] In some embodiments, the polymer P comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof. In some embodiments, the polymer P has the following general formula: [ka] (wherein 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, n is 8 or 14. In some embodiments, n is 14. In some embodiments, R1 and R2 in the above formula are -(CH2) 13 CH3 (myristyl) and the polymer P has the following general formula: [ka] (wherein n is 14) Includes.

[0230] In some embodiments, the targeting compound (the hydrophobic moiety to which the binding moiety is covalently attached) has the following general formula: [ka] wherein P, X2 and B are as defined above, and R t1 and R t2 each independently is H or methyl In some embodiments, R t1 and R t2 are both methyl. In some embodiments, R t1 is methyl and R t2 is H. In some embodiments, R t1 is H and R t2 is methyl. In some embodiments, R t1 and Rt2 are both H.

[0231] In some embodiments, the targeting compound (the hydrophobic moiety to which the binding moiety is covalently attached) has the following general formula: [ka] (wherein P, X2 and B are as defined above). Includes.

[0232] In some embodiments, the 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 a derivative thereof. In some embodiments, the polymer P has the following general formula: [ka] (wherein 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, n is 8 or 14. In some embodiments, n is 8. In some embodiments, n is 14.

[0233] In some embodiments, the targeting compound (the hydrophobic moiety to which the binding moiety is covalently attached) has the following general formula: [ka] wherein X1, P, X2, and B are as defined above, and R1 and R2 independently comprise an acyl moiety. In some embodiments, at least one, e.g., each acyl moiety is linear or branched, preferably linear. In some embodiments, at least one, e.g., each acyl moiety has at least 8 carbon atoms, e.g., 8 to 24, e.g., 10 to 18 carbon atoms. Preferably, at least one, e.g., each acyl moiety is an acyl moiety of a fatty acid, more preferably, at least one, e.g., each acyl moiety is an acyl moiety of a fatty acid having at least 8 carbon atoms, e.g., 8 to 24, e.g., 10 to 18 carbon atoms. Examples of acyl moieties include CH3(CH2) 16 C(O)-(Stearoyl), CH3(CH2) 14 C(O)-(palmitoyl), and CH3(CH2) 12 In some embodiments, both acyl groups are CH3(CH2) 16 C(O)-(stearoyl). In some embodiments, both acyl groups are CH(CH). 12 In some embodiments, X is absent or -HPO-(CH). n It contains —NH—, and n is 1 to 5, for example, 2.

[0234] In some embodiments, the polymer P comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof. In some embodiments, the polymer P has the following general formula: [ka] (wherein 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, n is 8 or 14. In some embodiments, n is 8. In some embodiments, n is 14.

[0235] In some embodiments, polymer P comprises pSar. In some embodiments, polymer P has the following general formula: [ka] (In the formula, 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.

[0236] In some embodiments, the targeting compound (the hydrophobic moiety to which the binding moiety is covalently attached) has the following general formula: [ka] wherein P, X2, and B are as defined above, and R1 and R2 independently comprise an acyl moiety. In some embodiments, at least one, e.g., each acyl moiety is linear or branched, preferably linear. In some embodiments, at least one, e.g., each acyl moiety has at least 8 carbon atoms, e.g., 8 to 24, e.g., 10 to 18 carbon atoms. Preferably, at least one, e.g., each acyl moiety is an acyl moiety of a fatty acid, more preferably, at least one, e.g., each acyl moiety is an acyl moiety of a fatty acid having at least 8 carbon atoms, e.g., 8 to 24, e.g., 10 to 18 carbon atoms. Examples of acyl moieties include CH3(CH2) 16 C(O)-(Stearoyl), CH3(CH2) 14 C(O)-(palmitoyl), and CH3(CH2) 12 In some embodiments, both acyl groups are CH3(CH2) 16 C(O)-(stearoyl). In some embodiments, both acyl groups are CH(CH). 12 C(O)-(myristoyl).

[0237] In some embodiments, the polymer P comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof. In some embodiments, the polymer P has the following general formula: [ka] (wherein 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, n is 8 or 14. In some embodiments, n is 8. In some embodiments, n is 14.

[0238] In some embodiments, n is 8 and R1 and R2 are CH3(CH2) 16 In some embodiments, n is 14 and R1 and R2 are CH3(CH2). 16 C(O)-(stearoyl).

[0239] In some embodiments, n is 8 and R1 and R2 are CH3(CH2) 12 In some embodiments, n is 14 and R1 and R2 are CH3(CH2). 12 C(O)-(myristoyl).

[0240] In some embodiments, polymer P comprises pSar. In some embodiments, polymer P has the following general formula: [ka] (In the formula, 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.

[0241] In some embodiments, s is 20 and R1 and R2 are CH3(CH2) 16 C(O)-(stearoyl).

[0242] In some embodiments, s is 20 and R1 and R2 are CH3(CH2) 12 C(O)-(myristoyl).

[0243] In some embodiments, X2 in the above formula comprises the reaction product of a thiol or cysteine ​​reactive group, e.g., a maleimide group, with a compound comprising a thiol or cysteine ​​group. In some embodiments, the compound comprising a thiol or cysteine ​​group has the formula SH(CH2) n C(O)—, where n is in the range of 1 to 5. In some embodiments, n is 2. In some embodiments, X2 comprises a thiosuccinimide moiety.

[0244] In some embodiments, X2 has the following general formula: [ka] Includes.

[0245] In some embodiments, X2 has the following general formula: [ka] (wherein 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.

[0246] In one aspect, the present disclosure provides a targeting compound as described herein. In some embodiments of the targeting compound, the binding moiety comprises a moiety that binds to a cell surface antigen, such as a primary targeting moiety as described herein. In some embodiments of the targeting compound, the binding moiety comprises a moiety that binds to a docking compound. In some embodiments of the targeting compound, the binding moiety comprises an epitope tag, such as an ALFA tag, such as an ALFA tag as described herein.

[0247] Thus, the present disclosure provides, in one aspect, a compound having the following general formula: L-X1-P-X2-B (In the formula, P comprises a polymer; L comprises a hydrophobic moiety (e.g., a lipid) attached to a first end of the polymer; B comprises a primary targeting moiety, as described herein, attached to the second terminus of the polymer; X1 is absent or a first linking moiety; and X2 is absent or a second linking moiety The present invention provides a compound of the formula:

[0248] In one aspect, the present disclosure provides a compound having the following general formula: L-X1-P-X2-B (In the formula, P comprises a polymer; L comprises a hydrophobic moiety (e.g., a lipid) attached to a first end of the polymer; B comprises an epitope tag, e.g., an ALFA tag, e.g., an ALFA tag described herein, attached to the second terminus of the polymer; X1 is absent or a first linking moiety; and X2 is absent or a second linking moiety Further provided is a compound of the formula:

[0249] In some embodiments, X1 comprises a carbonyl group. In some embodiments, L comprises a phosphatidylethanolamine, which can be linked to P by an amide group.

[0250] In some embodiments, X2 comprises the reaction product of a thiol- or cysteine-reactive group, e.g., a maleimide group, with a thiol- or cysteine ​​group of a compound comprising an epitope tag. In some embodiments, X2 comprises a thiosuccinimide moiety.

[0251] In some embodiments, L comprises a lipid as described above. In some embodiments, L comprises DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine), which can be linked to P by an amide group.

[0252] In some embodiments, P comprises a polymer as described above. In some embodiments, P comprises a polymer that provides stealth properties, extends circulating half-life, and / or reduces nonspecific protein binding or cell adhesion. In some embodiments, 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, P comprises polyethylene glycol (PEG); e.g., PEG as described above.

[0253] In some embodiments, L-X1-P comprises an amphiphilic derivative of the polymer described above. In some embodiments, the amphiphilic derivative of the polymer comprises a conjugate of disteroyl-glycero-phosphoethanolamine (DSPE) and a polymer, such as a polymer described above. In some embodiments, the amphiphilic derivative of the polymer comprises a disteroyl-glycero-phosphoethanolamine-polyethylene glycol conjugate (DSPE-PEG).

[0254] In some embodiments, the targeting compound can be obtained by reacting a thiol or cysteine ​​group of a compound comprising a primary targeting moiety or epitope tag with 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).

[0255] In some embodiments, the thiol or cysteine ​​reactive group comprises a maleimide group.

[0256] In some embodiments, the PEG reagent comprises DSPE-PEG-maleimide. In some embodiments, the compound comprising the primary targeting moiety or epitope tag has the formula SH(CH2) n C(O)-B, where n is in the range of 1 to 5, and B comprises a primary targeting moiety or epitope tag. In some embodiments, n is 2.

[0257] In some embodiments, the targeting compound comprises 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)] and a compound of formula SH(CH) n and a compound comprising C(O)-B, where n is in the range of 1 to 5, and B comprises a primary targeting moiety or epitope tag. In some embodiments, n is 2.

[0258] In some embodiments, the targeting compound (the hydrophobic moiety to which the binding moiety is covalently attached) has the following general formula: [ka] wherein B comprises an epitope tag, e.g., an ALFA tag, e.g., an ALFA tag described herein. Includes.

[0259] In some embodiments, the targeting compound (the hydrophobic moiety to which the binding moiety is covalently attached) has the following general formula: [ka] (In the formula, X2 is as defined above, and R1 and R2 are CH3(CH2) 16 C(O)-(stearoyl) or CH3(CH2) 12 C(O)-(myristoyl), and the polymer P has the following general formula: [ka] (In the formula, 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 B comprises an epitope tag, e.g., an ALFA tag, e.g., an ALFA tag described herein. (including Includes.

[0260] In some embodiments, n is 8 and R1 and R2 are CH3(CH2) 16 In some embodiments, n is 14 and R1 and R2 are CH3(CH2). 16 C(O)-(stearoyl).

[0261] In some embodiments, n is 8 and R1 and R2 are CH3(CH2) 12 In some embodiments, n is 14 and R1 and R2 are CH3(CH2). 12 C(O)-(myristoyl).

[0262] In some embodiments, X2 has the following general formula: [ka] Includes.

[0263] In some embodiments, the targeting compound (the hydrophobic moiety to which the binding moiety is covalently attached) has the following general formula: [ka] (In the formula, X2 is as defined above, and R1 and R2 are CH3(CH2) 16 C(O)-(stearoyl) or CH3(CH2) 12 C(O)-(myristoyl), and the polymer P has the following general formula: [ka] (In the formula, 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 B comprises an epitope tag, e.g., an ALFA tag, e.g., an ALFA tag described herein. (including Includes.

[0264] In some embodiments, s is 20 and R1 and R2 are CH3(CH2) 16 C(O)-(stearoyl).

[0265] In some embodiments, s is 20 and R1 and R2 are CH3(CH2) 12 C(O)-(myristoyl).

[0266] In some embodiments, X2 has the following general formula: [ka] Includes.

[0267] In some embodiments, B comprises a moiety comprising the structure -N-peptide-C(O)-NH2, wherein the peptide comprises an epitope tag, e.g., an ALFA tag, e.g., an ALFA tag described herein.

[0268] In one aspect, the present disclosure provides a targeting compound as described above incorporated into a particle (e.g., a particle described herein) via a hydrophobic component (e.g., a lipid component) of the targeting compound.

[0269] Primary targeting part In accordance with the present disclosure, nucleic acid payloads are specifically delivered to target cells by providing a moiety that binds to a target on the target cell, e.g., an antigen on the target cell, thereby targeting the particle containing the nucleic acid payload to the target cell.

[0270] In some embodiments, the moiety that binds to a target on a target cell is comprised by a compound (targeting compound) that is an integral part of the payload-bearing particle. In these embodiments, the targeting compound comprises a binding moiety that binds to a target cell.

[0271] In some embodiments, the moiety that binds to a target on a target cell is comprised by a compound (docking compound) that is an integral part of the payload-bearing particle and further comprises a moiety that binds to a compound (targeting compound) that comprises a moiety for binding to a docking compound. In these embodiments, the targeting compound itself preferably does not comprise a moiety that binds to a target on a target cell. Rather, the targeting compound comprises a binding moiety that forms a binding partner for the docking compound that binds to the target cell.

[0272] The target on the target cell is also referred to herein as the “primary target.” In some embodiments, the primary target is a cell surface antigen on the target cell.

[0273] As used herein, the term "primary targeting moiety" refers to the portion of a targeting compound or docking compound that binds to a primary target, such as a cell surface antigen on a target cell. Such targeting moieties are typically moieties that have affinity for cell surface targets. These moieties can be any peptide or protein (e.g., antibody or antibody fragment) that binds to the primary target. Specific embodiments of suitable primary targeting moieties for use herein include cell surface antigen-binding moieties, such as antibodies, antibody fragments, and DARPins. Another example of a primary targeting moiety is a peptide or protein that binds to a receptor.

[0274] The primary targeting moiety preferably binds with high specificity and / or high affinity, and the binding to the primary target is preferably stable in the body.

[0275] To enable specific targeting of the primary target, the primary targeting moiety of the targeting compound or docking compound can include compounds including, but not limited to, antibodies, antibody fragments, such as Fab2, Fab, scFV, VHH domains, and other proteins or peptides.

[0276] According to some embodiments, the primary target is a cell surface antigen, such as a T cell antigen, for example, CD3, e.g., CD3e, CD8 or CD4, and suitable primary targeting moieties include, but are not limited to, peptides and polypeptides that target cell surface antigens, such as antibodies, antibody fragments and DARPins.

[0277] According to some embodiments, the primary target is a receptor, and suitable primary targeting moieties include, but are not limited to, a ligand of such a receptor or a portion thereof that still binds to the receptor, e.g., a receptor-binding peptide in the case of a receptor-binding protein ligand.

[0278] Other examples of proteinaceous primary targeting moieties include interferons, such as alpha, beta and gamma interferon, interleukins, and protein growth factors, such as transforming growth factor (TGF) or platelet-derived growth factor (PDGF).

[0279] According to some embodiments, the primary target and the primary targeting moiety are selected to achieve specific or increased targeting of a certain cell.This can be achieved by selecting a primary target with cell-specific expression.For example, T cell antigens, such as those described herein, can be expressed in T cells, and they are not expressed or expressed at a lower level in other cells.

[0280] Docking Compounds In some embodiments, a "docking compound" is used to form a connection between a primary target, e.g., a target cell or an antigen on a target cell, and a targeting compound incorporated into a particle containing a nucleic acid payload to be delivered to the target cell. In some embodiments, the connection between the primary target, e.g., a target cell or an antigen on a target cell, and the docking compound is a non-covalent connection. In some embodiments, the connection between the docking compound and the targeting compound is a non-covalent or covalent connection. In some embodiments, the targeting compound includes a binding moiety for binding to the docking compound that is covalently attached to a hydrophobic moiety (e.g., a lipid). The hydrophobic moiety (e.g., a lipid) forms part of the particle.

[0281] In some embodiments, the docking compound comprises a "primary targeting moiety," e.g., a moiety that targets a primary target of interest, e.g., a cell surface antigen on a target cell, that has the ability to bind to the cell surface antigen on the target cell. In some embodiments, "primary targeting moiety," as used herein, refers to the portion of the docking compound that binds to the primary target.

[0282] The docking compound further comprises a group that serves as a binding partner for each binding moiety of the targeting compound. The portion of the targeting compound that comprises a hydrophobic portion (e.g., lipid) (to which the binding moiety for the docking compound is covalently attached) is incorporated into the particle carrying the payload, thereby forming a connection between the particle and the docking compound. The portion of the docking compound that binds to the targeting compound and the primary targeting moiety are preferably linked to each other by a covalent bond.

[0283] According to some embodiments, the docking compound comprises a bispecific molecule, e.g., a bispecific polypeptide, e.g., a bispecific antibody. In some embodiments, the docking compound comprises a binding domain that binds to a primary target and a binding domain that binds to a targeting compound. In some embodiments, the docking compound comprises an antibody or antibody fragment that binds to a primary target and an antibody or antibody fragment that binds to a targeting compound. In some embodiments, at least one binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. In some embodiments, each binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. In some embodiments, at least one binding domain comprises a single domain antibody, such as a VHH. In some embodiments, each binding domain comprises a single domain antibody, such as a VHH. In some embodiments, one binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody, and the other binding domain comprises a single domain antibody, such as a VHH. In some embodiments, the binding domain that binds to the primary target comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. In some embodiments, the binding domain that binds to the primary target comprises a single domain antibody, such as a VHH. In some embodiments, the binding domain that binds to the targeting compound comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. In some embodiments, the binding domain that binds to the targeting compound comprises a single domain antibody, such as a VHH.

[0284] In some embodiments, the docking compound comprises a fusion protein comprising a binding domain that binds to the primary target and a binding domain that binds to the targeted compound.

[0285] In some embodiments, the docking compound comprises a single peptide chain. In some embodiments, the single peptide chain comprises a portion that binds to a primary target, such as an antibody, an antibody fragment, or a DARPin, and a portion that binds to a targeting compound, such as an antibody or an antibody fragment. In some embodiments, the antibody fragment is a VHH, an scFv, or a mixture thereof. In different embodiments, the docking compound comprises one of the following structures (from N-terminus to C-terminus): VHH(α targeting compound)-optional linker-VHH(α primary target) VHH(α primary target)-optional linker-VHH(α targeting compound) VHH (α targeting compound) - optional linker - scFv (α primary target) scFv (α primary targeting) - optional linker - VHH (α targeting compound) VHH (α primary target) - optional linker - scFv (α targeting compound) scFv (α targeting compound) - optional linker - VHH (α primary target) scFv(α targeting compound)-optional linker-scFv(α primary target) scFv(α primary targeting)-optional linker-scFv(α targeting compound)

[0286] In one aspect, the present disclosure provides a docking compound described herein. In some embodiments, the docking compound comprises a bispecific molecule, e.g., a bispecific polypeptide, e.g., a bispecific antibody, where one specificity binds to an epitope tag, e.g., an ALFA tag, and the other specificity binds to a primary target, e.g., a cell surface antigen on a target cell. In some embodiments, the specificity that binds to the epitope tag is an antibody or antibody fragment, such as an NbALFA-nanobody (NbALFA). In some embodiments, the specificity that binds to the primary target is an antibody, antibody fragment, or DARPin. In some embodiments, the portion of the docking compound that targets the primary target is selected from the group consisting of an anti-primary target DARPin, an anti-primary target VHH, and an anti-primary target scFv, and / or the portion of the docking compound that binds to the targeting compound is an NbALFA-nanobody (NbALFA). In some embodiments, the docking compound has a structure selected from the group consisting of NbALFA x anti-primary target DARPin, NbALFA x anti-primary target VHH, and NbALFA x anti-primary target scFv. In some embodiments, the primary target is a T cell antigen, e.g., CD3, e.g., CD3e, CD4, or CD8. In some embodiments, the docking compound comprises a bispecific antibody comprising a nanobody that binds to an epitope tag, e.g., an ALFA tag, and an anti-CD3 VHH. In some embodiments, the docking compound comprises a bispecific antibody comprising a nanobody that binds to an epitope tag, e.g., an ALFA tag, and an anti-CD3 scFv. In some embodiments, the docking compound comprises a bispecific molecule comprising a nanobody that binds to an epitope tag, e.g., an ALFA tag, and an anti-CD3 DARPin. In some embodiments, the docking compound comprises a bispecific antibody comprising a nanobody that binds to an epitope tag, e.g., an ALFA tag, and an anti-CD4 VHH. In some embodiments, the docking compound comprises a bispecific antibody comprising a nanobody that binds to an epitope tag, e.g., an ALFA tag, and an anti-CD4 scFv.In some embodiments, the docking compound comprises a bispecific molecule comprising a nanobody that binds to an epitope tag, e.g., an ALFA tag, and an anti-CD4 DARPin. In some embodiments, the docking compound comprises a bispecific antibody comprising a nanobody that binds to an epitope tag, e.g., an ALFA tag, and an anti-CD8 VHH. In some embodiments, the docking compound comprises a bispecific antibody comprising a nanobody that binds to an epitope tag, e.g., an ALFA tag, and an anti-CD8 scFv. In some embodiments, the docking compound comprises a bispecific molecule comprising a nanobody that binds to an epitope tag, e.g., an ALFA tag, and an anti-CD8 DARPin.

[0287] Interactive moieties on the targeting compound and on the docking compound In some embodiments, the moieties on the targeting compound (the binding moieties covalently attached to the hydrophobic moieties) and the moieties on the docking compound (the moieties that bind to the binding moieties covalently attached to the hydrophobic moieties) that interact with each other are non-covalently bound to each other.

[0288] In some embodiments, the moieties on the targeting compound and the docking compound that interact with each other bind to each other under physiological conditions.

[0289] In some embodiments, the moieties on the targeting compound and the docking compound that interact with each other are an antibody / antigen system.

[0290] In some embodiments, the portion of the targeting compound that binds to the docking compound comprises a peptide or protein, e.g., a peptide tag, and the portion of the docking compound that binds to the targeting compound comprises a binding agent that binds to the peptide or protein, e.g., an antibody or antibody fragment.

[0291] In some embodiments, the portion of the docking compound that binds to the targeting compound comprises a peptide or protein, e.g., a peptide tag, and the portion of the targeting compound that binds to the docking compound comprises a binder that binds to the peptide or protein, e.g., an antibody or antibody fragment. In some embodiments, the portions on the targeting compound and the docking compound that interact with each other comprise an epitope tag / binder system.

[0292] As used herein, an "epitope tag" refers to a stretch of amino acids to which an antibody or a proteinaceous molecule with antibody-like function can bind.

[0293] In some embodiments, the epitope tag comprises an ALFA tag. In some embodiments, the epitope tag / binder system comprises an ALFA tag and an alpha-specific single domain antibody (sdAb), NbALFA-nanobody.

[0294] In some embodiments, the ALFA tag comprises the amino acid sequence -AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12-AA13-AA14-, Including, The amino acids AA0, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13 and AA14 are AA0 is Pro or deleted; AA1 is Ser, Gly, Thr, or Pro; AA2 is Arg, Gly, Ala, Glu, or Pro; AA3 is Leu, Ile, or Val; AA4 is Glu or Gln; AA5 is Glu or Gln; AA6 is Glu or Gln; AA7 is Leu, Ile, or Val; AA8 is Arg, Ala, Gln, or Glu; AA9 is Arg, Ala, Gln, or Glu; AA10 is Arg; AA11 is Leu; AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted; AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and AA14 is Pro or deleted.

[0295] In some embodiments, the ALFA tag comprises a sequence selected from the group consisting of SRLEEELRRRLTE, PSRLEEELRRRLTE, SRLEEELRRRLTEP, and PSRLEEELRRRLTEP.

[0296] In some embodiments, the ALFA tag comprises a circularized amino acid sequence -AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12-AA13-AA14-, Including, the side chains of any two (X1, X2) of the amino acids AAO, AAI, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AAIO, AA11, AA12, AA13, and AA14 are covalently connected; and The amino acids AA0, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13, and AA14 that are not X1 and X2 are AA0 is Pro or deleted; AA1 is Ser, Gly, Thr, or Pro; AA2 is Arg, Gly, Ala, Glu, or Pro; AA3 is Leu, Ile, or Val; AA4 is Glu or Gln; AA5 is Glu or Gln; AA6 is Glu or Gln; AA7 is Leu, Ile, or Val; AA8 is Arg, Ala, Gln, or Glu; AA9 is Arg, Ala, Gln, or Glu; AA10 is Arg; AA11 is Leu; AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted; AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and AA14 is Pro or deleted.

[0297] In some embodiments, X1 and X2 are separated by two or three amino acids.

[0298] In some embodiments, AA5 is X1 and AA9 is X2, or AA5 is X1 and AA8 is X2, or AA9 is X1 and AA13 is X2, or AA6 is X1 and AA9 is X2, or AA9 is X1 and AA12 is X2, or AAIO is X1 and AA13 is X2, or AA6 is X1 and AAIO is X2, or AA4 is X1 and AA8 is X2.

[0299] In some embodiments, the ALFA tag is a. -AA0-AA1-AA2-AA3-AA4-cyclo(X1-AA6-AA7-AA8-X2)-Arg-Leu-AA12-AA13-AA14-, b. -AA0-AA1-AA2-AA3-AA4-cyclo(X1-AA6-AA7-X2)-AA9-Arg-Leu-AA12-AA13-AA14-, c. -AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-cyclo(X1-Arg-Leu-AA12-X2)-AA14-, d. -AA0-AA1-AA2-AA3-AA4-AA5-cyclo(X1-AA7-AA8-X2)-Arg-Leu-AA12-AA13-AA14-, e. -AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-cyclo(X1-Arg-Leu-X2)-AA13-AA14-, f. -AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-cyclo(X1-Leu-AA12-X2)-AA14-, g. -AA0-AA1-AA2-AA3-AA4-AA5-cyclo(X1-AA7-AA8-AA9-X2)-Leu-AA12-AA13-AA14-, and h. -AA0-AA1-AA2-AA3-cyclo(X1-AA5-AA6-AA7-X2)-AA9-Arg-Leu-AA12-AA13-AA14- and a cyclic amino acid sequence selected from the group consisting of: the side chains of the X1 and X2 amino acid residues are covalently connected; AA0 is Pro or deleted; AA1 is Ser, Gly, Thr, or Pro; AA2 is Arg, Gly, Ala, Glu, or Pro; AA3 is Leu, Ile, or Val; AA4 is Glu or Gln; AA5 is Glu or Gln; AA6 is Glu or Gln; AA7 is Leu, Ile, or Val; AA8 is Arg, Ala, Gln, or Glu; AA9 is Arg, Ala, Gln, or Glu; AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted; AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and AA14 is Pro or deleted.

[0300] In some embodiments, X1 and X2 in the peptides disclosed herein are covalently linked via an amide, disulfide, thioether, ether, ester, thioester, thioamide, alkylene, alkenylene, alkynylene, and / or 1,2,3-triazole.

[0301] In some embodiments, the cyclized amino acid sequences described herein are generated by linking the amino group on the side chain of one of X1 and X2 to the carboxyl group on the side chain of the other of X1 and X2 via an amide bond. The amino group on the side chain of an amino acid having a pendant amine group, e.g., lysine or a lysine derivative, and the carboxyl group on the side chain of an acidic amino acid, e.g., aspartic acid, glutamic acid, or a derivative thereof, can be used to generate cyclized amino acid sequences via an amide bond.

[0302] In some embodiments, the cyclized amino acid sequences described herein are generated by linking a sulfhydryl group on the side chain of one of X1 and X2 to a sulfhydryl group on the side chain of the other of X1 and X2 via a disulfide bond. Sulfhydryl-containing amino acids include cysteine ​​and other sulfhydryl-containing amino acids as Pen.

[0303] In some embodiments, 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, with the proviso that 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; when X1 is Cys, DCys, hCys, DhCys, Pen, or DPen, X2 is Cys, DCys, hCys, DhCys, Pen, or DPen.

[0304] In some embodiments, 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- has the following structure: [ka] Includes.

[0305] In some embodiments, X1 is Lys and X2 is Glu. In some embodiments, -cyclo(Lys------Glu)-, -c(Lys------Glu)-, -cyclo(K------E)-, -c(K-------E)-, -KE-cyclo, or cycloK------cycloE- has the following structure: [ka] Includes.

[0306] In some embodiments, X1 is Cys and X2 is Cys. In some embodiments, -cyclo(Cys------Cys)-, c(Cys------Cys)-, -cyclo(C------C)-, -c(C--------C)-, -C----C-cyclo, or -cycloC----cycloC- has the following structure: [ka] Includes.

[0307] Specific cyclized amino acid sequences of the general formula identified above include, for example: -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Glu)-, -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Asp)-, -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-シクロ(DGlu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Glu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-シクロ(Glu-Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Cys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Cys-Arg-Leu-Thr-Cys)-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-Cys)-、 -Pro-Ser-Arg-Leu-Glu-Glu-シクロ(Cys-Leu-Arg-Arg-Cys)-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Cys-Arg-Leu-Thr-Cys)-、 -Ser-Arg-Leu-Glu-シクロ(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-Cys)-、 -Ser-Arg-Leu-Glu-Glu-シクロ(Cys-Leu-Arg-Arg-Cys)-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(DGlu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-Arg-DGlu)-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-Arg-DGlu)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DGlu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-Arg-DGlu)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-Arg-DGlu)-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(DAsp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(DAsp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(Asp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DAsp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DAsp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Asp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(DGlu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(Glu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(DGlu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-Glu)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-DGlu)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-DGlu)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(Asp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(DAsp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(DAsp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Glu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DGlu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu- -Pro-Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-Glu)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-DGlu)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-DGlu)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Asp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DAsp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DAsp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-Glu-シクロ(Asp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-Glu-シクロ(Lys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-Glu-シクロ(DAsp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-Glu-シクロ(Lys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-Glu-シクロ(Asp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-Glu-シクロ(DLys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-Glu-シクロ(DLys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-Glu-シクロ(DAsp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-シクロ(Asp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-シクロ(Lys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-シクロ(DAsp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-シクロ(Lys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-シクロ(Asp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-シクロ(DLys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-シクロ(DLys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-シクロ(DAsp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-Cys)-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Cys-Arg-Leu-Cys)-Glu-、 -Ser-Arg-Leu-Glu-Glu-シクロ(Cys-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-Cys)-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Cys-Arg-Leu-Cys)-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-シクロ(Cys-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(DCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(DCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(hCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(hCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(Cys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(hCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(DCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(hCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(DhCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(Cys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(DCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(DhCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(DhCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(hCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(hCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Cys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(hCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(hCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DhCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Cys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DhCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DhCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-DCys)-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DCys-Leu-Thr-Cys)-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DCys-Leu-Thr-DCys)-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(hCys-Leu-Thr-Cys)-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(hCys-Leu-Thr-DCys)-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(hCys-Leu-Thr-hCys)-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-hCys)-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DCys-Leu-Thr-hCys)-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-hCys)-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DhCys-Leu-Thr-Cys)-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DhCys-Leu-Thr-DCys)-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DhCys-Leu-Thr-hCys)-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-DhCys)-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DCys-Leu-Thr-DhCys)-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-DhCys)-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Pen-Leu-Thr-Pen)-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Pen-Leu-Thr-DPen)-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DPen-Leu-Thr-Pen)-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DPen-Leu-Thr-DPen)-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(DCys-Arg-Leu-Cys)-Glu-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(DCys-Arg-Leu-DCys)-Glu-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Cys-Arg-Leu-DCys)-Glu-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(hCys-Arg-Leu-Cys)-Glu-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(hCys-Arg-Leu-DCys)-Glu-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Cys-Arg-Leu-hCys)-Glu-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(hCys-Arg-Leu-hCys)-Glu-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(hCys-Arg-Leu-DhCys)-Glu-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(DhCys-Arg-Leu-hCys)-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-DCys)-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DCys-Leu-Thr-Cys)-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DCys-Leu-Thr-DCys)-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(hCys-Leu-Thr-Cys)-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(hCys-Leu-Thr-DCys)-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(hCys-Leu-Thr-hCys)-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-hCys)-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DCys-Leu-Thr-hCys)-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-hCys)-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DhCys-Leu-Thr-Cys)-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DhCys-Leu-Thr-DCys)-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DhCys-Leu-Thr-hCys)-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-DhCys)-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DCys-Leu-Thr-DhCys)-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-DhCys)-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Pen-Leu-Thr-Pen)-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Pen-Leu-Thr-DPen)-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DPen-Leu-Thr-Pen)-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DPen-Leu-Thr-DPen)-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(DCys-Arg-Leu-Cys)-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(DCys-Arg-Leu-DCys)-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Cys-Arg-Leu-DCys)-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(hCys-Arg-Leu-Cys)-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(hCys-Arg-Leu-DCys)-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Cys-Arg-Leu-hCys)-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(hCys-Arg-Leu-hCys)-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(hCys-Arg-Leu-DhCys)-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(DhCys-Arg-Leu-hCys)-Glu-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Lys-Arg-Leu-Asp)-Glu-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Asp-Arg-Leu-Lys)-Glu-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Lys-Arg-Leu-Glu)-Glu-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Glu-Arg-Leu-Lys)-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Lys-Arg-Leu-Asp)-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Asp-Arg-Leu-Lys)-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Lys-Arg-Leu-Glu)-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Glu-Arg-Leu-Lys)-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Cys-Arg-Leu-Thr-Cys)-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-Cys)-、 -Pro-Ser-Arg-Leu-Glu-Glu-シクロ(Cys-Leu-Arg-Arg-Cys)-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(DGlu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-Arg-DGlu)-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-Arg-DGlu)-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DGlu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-Arg-DGlu)-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-Arg-DGlu)-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(DAsp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(DAsp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(Asp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DAsp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DAsp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Asp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(DGlu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(Glu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(DGlu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-Glu)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-DGlu)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-DGlu)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(Asp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(DAsp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(DAsp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Glu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DGlu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-Glu)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-DGlu)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-DGlu)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Asp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DAsp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DAsp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DLys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-Glu-シクロ(Asp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-Glu-シクロ(Lys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-Glu-シクロ(DAsp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-Glu-シクロ(Lys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-Glu-シクロ(Asp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-Glu-シクロ(DLys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-Glu-シクロ(DLys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-Glu-シクロ(DAsp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-シクロ(Asp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-シクロ(Lys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-シクロ(DAsp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-シクロ(Lys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-シクロ(Asp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-シクロ(DLys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-シクロ(DLys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-シクロ(DAsp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-Cys)-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Cys-Arg-Leu-Cys)-Glu-Pro-、 -Ser-Arg-Leu-Glu-Glu-シクロ(Cys-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-Cys)-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Cys-Arg-Leu-Cys)-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-シクロ(Cys-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(DCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(DCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(hCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(hCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(Cys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(hCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(DCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(hCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(DhCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(Cys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(DCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(DhCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-シクロ(DhCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(hCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(hCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Cys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(hCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(hCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DhCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Cys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DhCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DhCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-DCys)-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DCys-Leu-Thr-Cys)-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DCys-Leu-Thr-DCys)-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(hCys-Leu-Thr-Cys)-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(hCys-Leu-Thr-DCys)-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(hCys-Leu-Thr-hCys)-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-hCys)-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DCys-Leu-Thr-hCys)-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-hCys)-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DhCys-Leu-Thr-Cys)-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DhCys-Leu-Thr-DCys)-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DhCys-Leu-Thr-hCys)-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-DhCys)-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DCys-Leu-Thr-DhCys)-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-DhCys)-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Pen-Leu-Thr-Pen)-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Pen-Leu-Thr-DPen)-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DPen-Leu-Thr-Pen)-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DPen-Leu-Thr-DPen)-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(DCys-Arg-Leu-Cys)-Glu-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(DCys-Arg-Leu-DCys)-Glu-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Cys-Arg-Leu-DCys)-Glu-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(hCys-Arg-Leu-Cys)-Glu-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(hCys-Arg-Leu-DCys)-Glu-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Cys-Arg-Leu-hCys)-Glu-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(hCys-Arg-Leu-hCys)-Glu-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(hCys-Arg-Leu-DhCys)-Glu-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(DhCys-Arg-Leu-hCys)-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-DCys)-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DCys-Leu-Thr-Cys)-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DCys-Leu-Thr-DCys)-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(hCys-Leu-Thr-Cys)-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(hCys-Leu-Thr-DCys)-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(hCys-Leu-Thr-hCys)-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-hCys)-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DCys-Leu-Thr-hCys)-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-hCys)-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DhCys-Leu-Thr-Cys)-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DhCys-Leu-Thr-DCys)-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DhCys-Leu-Thr-hCys)-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-DhCys)-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DCys-Leu-Thr-DhCys)-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-DhCys)-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Pen-Leu-Thr-Pen)-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Pen-Leu-Thr-DPen)-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DPen-Leu-Thr-Pen)-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(DPen-Leu-Thr-DPen)-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(DCys-Arg-Leu-Cys)-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(DCys-Arg-Leu-DCys)-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Cys-Arg-Leu-DCys)-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(hCys-Arg-Leu-Cys)-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(hCys-Arg-Leu-DCys)-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Cys-Arg-Leu-hCys)-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(hCys-Arg-Leu-hCys)-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(hCys-Arg-Leu-DhCys)-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(DhCys-Arg-Leu-hCys)-Glu-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Lys-Arg-Leu-Asp)-Glu-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Asp-Arg-Leu-Lys)-Glu-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Lys-Arg-Leu-Glu)-Glu-Pro-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Glu-Arg-Leu-Lys)-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Lys-Arg-Leu-Asp)-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Asp-Arg-Leu-Lys)-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Lys-Arg-Leu-Glu)-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Glu-Arg-Leu-Lys)-Glu-Pro-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Thr-Cys)-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Cyclo(Cys-Leu-Arg-Arg-Cys)-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-, and -Ser-Arg-Leu-cyclo(Glu-Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-.

[0308] In some embodiments, the cyclic peptide is attached to the 3-mercaptopropionyl moiety through the α-amine moiety of the left-most amino acid in the cyclic peptide. In some embodiments, the right-most amino acid in the cyclic peptide comprises an amide.

[0309] In some embodiments, the circularized amino acid sequence is selected from the group consisting of: -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Glu)-, -Ser-Arg-Leu-Glu-シクロ(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-シクロ(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-、 -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Lys-Arg-Leu-Thr-Asp)-、 -Ser-Arg-Leu-Glu-シクロ(Glu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu- -Pro-Ser-Arg-Leu-Glu-シクロ(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(DGlu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu- -Pro-Ser-Arg-Leu-Glu-シクロ(Glu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu- -Pro-Ser-Arg-Leu-Glu-シクロ(Lys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu- -Pro-Ser-Arg-Leu-シクロ(Glu-Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Cys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-シクロ(Cys-Arg-Leu-Thr-Cys)-、 -Pro-Ser-Arg-Leu-Glu-シクロ(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-、 -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-シクロ(Cys-Leu-Thr-Cys)-、 -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Arg-Cys)-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Thr-Cys)-, -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-, -Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Arg-Cys)-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-, and -Ser-Arg-Leu-cyclo(Glu-Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-.

[0310] In some embodiments, the cyclic amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-. In some other embodiments, the cyclic amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-. In some still other embodiments, the cyclic amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-. In some still other embodiments, the cyclic amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-. In some still other embodiments, the cyclic amino acid sequence is -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Glu)-.

[0311] Cyclic peptides can have different cyclic bridge-forming moieties that form ring structures. Preferably, the ring structure contains a chemically stable bridge-forming moiety, such as an amide group, a lactone group, an ether group, a thioether group, a disulfide group, an alkylene group, an alkenyl group, or a 1,2,3-triazole group. The following are examples showing various bridge-forming moieties in peptides: [ka]

[0312] In some embodiments, the ALFA tag binding moiety comprises an antibody or antibody fragment, e.g., a camelid VHH domain. In some embodiments, the ALFA tag binding moiety comprises a single domain antibody (sdAb), NbALFA-nanobody.

[0313] In some embodiments, the ALFA tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence VTX1SALNAMAMG (where X1 is I or V), the CDR2 sequence AVSX2RGNAM (where X2 is E, H, N, D, or S), and the CDR3 sequence LEDRVDSFHDY.

[0314] In some embodiments, the ALFA tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence GVTX1SALNAMAMG (where X1 is I or V), the CDR2 sequence AVSX2RGNAM (where X2 is E, H, N, D, or S), and the CDR3 sequence LEDRVDSFHDY.

[0315] In some embodiments, the ALFA tag binding moiety comprises a single domain antibody, for example a camelid VHH domain comprising the CDR1 sequence VTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY.

[0316] In some embodiments, the ALFA tag binding moiety comprises a single domain antibody, for example a camelid VHH domain comprising the CDR1 sequence GVTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY.

[0317] In some embodiments, the ALFA tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the amino acid sequence EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity thereto, or a fragment of said amino acid sequence or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity thereto. In some embodiments, the amino acid sequence comprises the CDR1, CDR2, and CDR3 sequences described above.

[0318] In some embodiments, the epitope tag / binder system comprises an epitope tag (Spot tag) comprising the sequence PDRVRAVSHWSS, and the binder comprises a single domain antibody (sdAb, or nanobody) that specifically binds to the Spot tag (Spot-nanobody (14.7 kD)).

[0319] In some embodiments, after the moieties on the targeting compound and the docking compound that interact with each other are combined, a covalent bond is formed.In these embodiments, the system used herein can include a tag / catcher system that forms a covalent bond, such as SpyTag / SpyCatcher that forms an isopeptide bond.

[0320] The SpyTag / SpyCatcher system is a technology for irreversible conjugation of recombinant proteins. The peptide SpyTag spontaneously reacts with the protein SpyCatcher, forming an intermolecular isopeptide bond between the pair. Using the tag / catcher pair, bioconjugation can be achieved between two recombinant proteins.

[0321] In one embodiment, the present disclosure provides a conjugate in which a particle comprising a targeting compound (a hydrophobic moiety to which a binding moiety is covalently attached) is bound to a docking compound (a compound comprising (i) a moiety that binds to the binding moiety covalently attached to the hydrophobic moiety and (ii) a moiety that targets a cell surface antigen), such that the targeting compound and the docking compound comprise moieties that interact with each other.

[0322] Thus, the present disclosure provides, in one aspect, (a) a particle comprising a hydrophobic moiety to which a binding moiety is covalently attached; and (b) a compound comprising (i) a moiety that binds to a binding moiety covalently attached to a hydrophobic moiety and (ii) a moiety that targets a cell surface antigen. The present invention provides a composite comprising:

[0323] Different embodiments of the conjugated targeting compound and docking compound are described herein.

[0324] In some embodiments, the targeting compound comprises an ALFA tag. In these embodiments, the moiety of the docking compound that binds to the targeting compound can be an NbALFA-nanobody (NbALFA). In some embodiments, the docking compound can have a structure selected from the group consisting of NbALFA x anti-primary target DARPin, NbALFA x anti-primary target VHH, and NbALFA x anti-primary target scFv.

[0325] Targeting Compound Embodiments In some embodiments, the targeted compounds described herein have the structure (I): [ka] wherein the lipid is a phospholipid attached to a carbonyl through the amino group of its ethanolamine moiety, the PEG has a molecular weight of about 130 to about 50,000, the α-amino group of the left-most amino acid group of the peptide is attached to the carbonyl group of the 3-mercaptopropionyl moiety, and the peptide contains about 11 to about 15 amino acids, and Z is a bond or -CH-. In some embodiments, the lipid-PEG-peptide conjugate compound has the following structure (II): [ka] It has.

[0326] A particular embodiment of a lipid-PEG-peptide conjugate compound having structure (I) or (II) is where Z is a bond. In one embodiment, the peptide has the sequence -SRLEEELRRRLTE-. In another embodiment, the peptide has the sequence -PSRLEEELRRRLTE-. In other embodiments, the peptide is -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-L eu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Glu)-, -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Th r-Glu-, -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Lys Lo(Lys-Arg-Leu-Thr-Asp)-, -Pro-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-L eu-Glu-cyclo(DGlu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-DLys)-Arg- Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-cyclo(Glu- Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-,A cyclic peptide selected from the group consisting of -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Thr-Cys)-, -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-, and -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Arg-Cys)-Leu-Thr-Glu-.

[0327] The term "PEG" as used in Formula I above refers to any polyethylene glycol or other polyalkylene ether polymer. In one embodiment, PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In one embodiment, PEG is unsubstituted. In one embodiment, PEG is substituted, for example, with one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In one embodiment, 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 6000, in another embodiment, about 150 to about 5000, in another embodiment, about 150 to about 4000, in another embodiment, about 150 to about 3000, in another embodiment, about 300 to about 3000, in another embodiment, about 1000 to about 3000, and in yet another embodiment, about 1500 to about 2500.

[0328] In certain embodiments, the PEG (conjugated to the lipid) comprises "PEG2k," also referred to as "PEG2000," having an average molecular weight of about 2000 daltons. Another name for a lipid comprising polyethylene glycol PEG, such as PEG2K, is a "pegylated lipid," and when a phosphorus-containing linkage is present, the lipid is generally referred to herein as a "phospholipid" or "pegylated phospholipid."

[0329] In some embodiments, the lipid moiety to which PEG is attached in the functionalized stealth lipid disclosed in Formula I above comprises a neutral phospholipid. Examples of neutral phospholipids include dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoyl-sn-glycero-3-phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1-myristoyl-2-stearoylphosphatidylcholine (PSPC), 1-myristoyl-2-stearoylphosphatidylcholine (MPPC), 1-myristoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1-myris ... Lipids include, but are not limited to, 2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof.In certain embodiments, the preferred lipid is distearoyl-phosphatidylethanolamine (DSPE).

[0330] The terms "PEPTIDE" or "Peptide" are used interchangeably and, when used in Formula I or II above, refer to a series of amino acids connected sequentially by peptide bonds between the amino and carboxy groups of adjacent residues. "Peptide-NH" indicates that the C-terminal carboxyl group of the peptide is an amide. In some embodiments, the term "peptide" or "peptide" refers to an epitope tag, e.g., an ALFA tag, such as the ALFA tags described herein.

[0331] Particles containing nucleic acid payloads The particles described herein include one or more particles forming a drug, a nucleic acid payload to be delivered to a target cell, and a hydrophobic portion to which a binding moiety for binding to a target cell or a docking compound is covalently attached.

[0332] Nucleic Acid Payload In some embodiments, the nucleic acid payload, e.g., the nucleic acid payload that is delivered to a target cell to genetically modify the target cell and enable the target cell to express a biomolecule, e.g., a peptide or protein, encoded by the nucleic acid, comprises DNA, RNA, or a mixture thereof.

[0333] In some embodiments, the agents and methods described herein are used for targeted therapy, which can be achieved by using a payload containing one or more nucleic acids that are pharmaceutically active agents.

[0334] The term "pharmaceutically active agent" relates to any agent, such as a compound or cell, that is therapeutically effective when administered to an individual. The term "pharmaceutically active agent" further relates to any agent that modifies, preferably cures, alleviates or partially arrests the clinical symptoms of a given disease and its complications in a therapeutic intervention that involves the administration of the agent.

[0335] In some embodiments, the pharmaceutically active agent comprises a pharmaceutically active nucleic acid, such as a pharmaceutically active RNA.

[0336] " Pharmaceutically active nucleic acid " is a nucleic acid, such as RNA, that encodes a pharmaceutically active peptide or protein, or is itself pharmaceutically active, for example, has one or more pharmacological activities as described for pharmaceutically active protein.For example, RNA can be one or more strands of RNA interference (RNAi).Such drugs include small interfering RNA (siRNA), or short hairpin RNA (shRNA), or the precursor of siRNA or microRNA-like RNA, that targets target transcript, for example, transcript that is related to the target endogenous disease.

[0337] A "pharmaceutically active peptide or protein" when provided to a subject in a therapeutically effective amount has a positive or beneficial effect on the subject's condition or pathology. Preferably, a pharmaceutically active peptide or protein has curative or palliative properties and can be administered to improve, alleviate, relieve, reverse, delay the onset, or reduce the severity of one or more symptoms of a disease or disorder. A pharmaceutically active peptide or protein can have preventative properties and can be used to delay the onset of a disease or reduce the severity of such a disease or pathological condition. The term "pharmaceutically active peptide or protein" includes whole proteins or polypeptides and can also refer to pharmaceutically active fragments thereof. The term can also include pharmaceutically active analogs of peptides or proteins. The term "pharmaceutically active peptide or protein" includes peptides and proteins that are antigens, i.e., administration of the peptide or protein to a subject elicits an immune response in the subject that can be therapeutic or partially or fully protective.

[0338] Examples of pharmaceutically active proteins include, but are not limited to, cytokines and immune system proteins, such as immunologically active compounds (e.g., interleukins, colony-stimulating factors (CSFs), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferons, integrins, addressins, serotiny receptors, T cell receptors, immunoglobulins, soluble major histocompatibility complex antigens, immunologically active compounds (e.g., soluble erythropoietin, ... Active antigens, e.g., bacterial, parasitic or viral antigens, allergens, autoantigens, antibodies), hormones (e.g., insulin, thyroid hormones, catecholamines, gonadotrophins, stimulating hormones, prolactin, oxytocin, dopamine, bovine somatotropin, leptin, etc.), growth hormones (e.g., human growth hormone), growth factors (e.g., epidermal growth factor, nerve growth factor, insulin-like growth factor, etc.), growth factor receptors, enzymes (e.g., tissue plasminogen activator, streptokinase, cholesterol biosynthesis or These include degradative enzymes, steroidogenic enzymes, kinases, phosphodiesterases, methylases, demethylases, dehydrogenases, cellulases, proteases, lipases, phospholipases, aromatases, cytochromes, adenylate or guanylate cyclases, neuramidases, etc.), receptors (steroid hormone receptors, peptide receptors), binding proteins (growth hormone or growth factor binding proteins, etc.), transcription and translation factors, tumor growth suppressor proteins (e.g., angiogenesis inhibitors, etc.), and Inhibitory proteins), structural proteins (such as collagen, fibroin, fibrinogen, elastin, tubulin, actin, and myosin), blood proteins (such as thrombin, serum albumin, factor VII, factor VIII, insulin, factor IX, factor X, tissue plasminogen activator, protein C, von Willebrand factor, antithrombin III, glucocerebrosidase, erythropoietin, granulocyte colony-stimulating factor (GCSF) or modified factor VIII, anticoagulants, and the like.

[0339] In some embodiments, the pharmaceutically active protein is a cytokine involved in regulating lymphoid homeostasis, preferably a cytokine involved in, and preferably inducing or enhancing, the development, priming, expansion, differentiation, and / or survival of T cells. In some embodiments, the cytokine is an interleukin. In some embodiments, the pharmaceutically active protein according to the present disclosure is an interleukin selected from the group consisting of IL-2, IL-7, IL-12, IL-15, and IL-21.

[0340] In some embodiments, the nucleic acid delivered to the target cell is a nucleic acid encoding an antigen receptor. In these embodiments, the target cell may be an immune cell or an immune effector cell.

[0341] particle Nucleic acid payloads can be administered with one or more delivery vehicles that protect the payload from degradation, maximize delivery to on-target cells, and minimize exposure to off-target cells.Such delivery vehicles can be complexed with or encapsulate the payload, and include a wide range of substances, including polymers, lipids, and their mixtures.In some embodiments, such delivery vehicles can form particles together with the payload.

[0342] In the context of the present disclosure, the term "particle" refers to a molecule or molecular complex, particularly a structured entity formed by a particle-forming compound. In some embodiments, the particle is a nucleic acid-containing particle, such as a particle containing DNA, RNA, or a mixture thereof. In some embodiments, the particle contains an envelope (e.g., one or more layers or lamellae) made of one or more amphiphilic substances (e.g., amphiphilic lipids). In this context, the term "amphiphilic substance" means that the substance has both hydrophilic and lipophilic properties. The envelope may also contain additional substances (e.g., additional lipids) that do not necessarily have amphiphilic properties. Thus, the particle may be a monolamellar or multilamellar structure in which the substance constituting one or more layers or lamellae contains one or more amphiphilic substances (e.g., selected from the group consisting of amphiphilic lipids), optionally in combination with additional substances (e.g., additional lipids) that do not necessarily have amphiphilic properties. In some embodiments, the term "particle" refers to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure. According to the present disclosure, the term "particle" includes nanoparticles.

[0343] The term "nanoparticle" refers to a nano-sized particle comprising at least one particle-forming agent, such as at least one cationic or cationic ionizable lipid, wherein all three external dimensions of the particle are nanoscale, i.e., at least about 1 nm and less than about 1000 nm. Preferably, the size of a particle is its diameter.

[0344] In some embodiments, the particles described herein are in the range of about 10 to about 2000 nm, e.g., 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 up to about 1900 nm (e.g., up to about 1800 nm, up to about 1700 nm, up to about 1600 nm, up to about 1500 nm, up to about 1400 nm, up to about 1300 nm, up to about 1200 nm, up to about 1100 nm, at most about 1000 nm, at most about 950 nm, at most about 900 nm, at most about 850 nm, at most about 800 nm, at most about 750 nm, at most about 700 nm, at most about 650 nm, at most about 600 nm, at most about 550 nm, or at most about 500 nm), for example, about 20 to about 1500 nm, for example, about 30 to about 1200 nm, about 40 to about 1100 nm, about 50 to about 1000 nm, about 60 to about 900 nm In some embodiments, the particles described herein have a size (e.g., diameter) in the range of about 40 nm to about 200 nm, e.g., 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.

[0345] In some embodiments, the particles described herein may be from about 50 nm to about 1000 nm, from about 50 nm to about 800 nm, from about 50 nm to about 700 nm, from about 50 nm to about 600 nm, from about 50 nm to about 500 nm, from about 50 nm to about 450 nm, from about 50 nm to about 400 nm, from about 50 nm to about 350 nm, from about 50 nm to about 300 nm, from about 50 nm to about 250 nm, nm, about 50nm to about 200nm, about 100nm to about 1000nm, about 100nm to about 800nm, about 100nm to about 700nm, about 100nm to about 600nm, about 100nm to about 5 00nm, approximately 100nm ~ approximately 450nm, approximately 100nm ~ approximately 400nm, approximately 100nm ~ approximately 350nm, approximately 100nm ~ approximately 300nm, approximately 100nm ~ approximately 250nm, approximately 100nm ~ Approximately 200nm, approximately 150nm to approximately 1000nm, approximately 150nm to approximately 800nm, approximately 150nm to approximately 700nm, approximately 150nm to approximately 600nm, approximately 150nm to approximately 500nm, approximately 150nm nm~about 450nm, about 150nm~about 400nm, about 150nm~about 350nm, about 150nm~about 300nm, about 150nm~about 250nm, about 150nm~about 200nm, about 2 In some embodiments, the particles described herein have an average diameter in the range of about 40 nm to about 200 nm, e.g., about 50 nm to about 180 nm, about 60 nm to about 160 nm, about 80 nm to about 150 nm, about 100 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, or about 80 nm to about 150 nm. In some embodiments, the particles described herein have an average diameter in the range of about 40 nm to about 200 nm, e.g., 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.

[0346] The particles described herein may exhibit a polydispersity index (PDI) of less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1, or less than about 0.05. By way of example, the particles may exhibit a polydispersity index ranging from about 0.01 to about 0.4 or from about 0.1 to about 0.3.

[0347] "Nucleic acid particles" can be used to deliver nucleic acids to a desired target site (e.g., a cell, tissue, organ, etc.). Nucleic acid particles can be formed from at least one cationic or cationic ionizable compound, such as a polymer or lipid, that complexes the nucleic acid. Without intending to be bound by any theory, it is believed that the cationic or cationic ionizable compound forms aggregates together with the nucleic acid, and this aggregation results in colloidally stable particles.

[0348] In some embodiments, the nucleic acid may be non-covalently associated with the particle, hi some embodiments, the nucleic acid may be attached to the outer surface of the particle (surface nucleic acid) and / or contained within the particle (encapsulated nucleic acid).

[0349] 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. This correlates with the charge ratio, since nitrogen atoms are usually positively charged (depending on the pH) and phosphate groups are negatively charged. If charge balance exists, the N / P ratio is pH dependent. Because positively charged nanoparticles are thought to favor transfection, lipid formulations are often formed with an N / P ratio greater than 4 and up to 12. In that case, the nucleic acid is considered fully bound to the nanoparticles.

[0350] The particles described herein can be prepared by a wide range of methods.For example, the method of preparing nucleic acid particles can include obtaining colloid from at least one cationic or cationically ionizable lipid, and mixing the colloid with nucleic acid to obtain nucleic acid particles.

[0351] The term "colloid," as used herein, refers to a type of homogeneous mixture in which dispersed particles do not settle. The insoluble particles in the mixture are microscopic, with particle sizes ranging from 1 to 1000 nanometers. The mixture may be called a colloid or a colloidal suspension. The term "colloid" may refer only to the particles in the mixture and not to the suspension as a whole.

[0352] For the preparation of colloids containing at least one cationic or cationic-ionizable lipid, suitably adapted methods conventionally used to prepare liposome vesicles are applicable herein. The most commonly used methods for preparing liposome vesicles share the following basic steps: (i) dissolving the lipid in an organic solvent, (ii) drying the resulting solution, and (iii) hydrating the dried lipid (using various aqueous media).

[0353] In the film hydration method, lipids are first dissolved in a suitable organic solvent and dried to obtain a thin film at the bottom of a flask. The resulting lipid film is hydrated with a suitable aqueous medium to produce a liposome dispersion. Further miniaturization steps may also be included.

[0354] Reverse phase evaporation is an alternative method to membrane hydration for preparing liposome vesicles, involving the formation of a water-in-oil emulsion between an aqueous phase and a lipid-containing organic phase. Brief sonication of this mixture is necessary to homogenize the system. Removal of the organic phase under reduced pressure results in a milky gel that then transforms into a liposome suspension.

[0355] The term "ethanol injection technique" refers to the process of rapidly injecting an ethanol solution containing lipids into an aqueous solution through a needle. This action disperses the lipids throughout the solution and promotes lipid structure formation, e.g., lipid vesicle formation, such as liposome formation. Generally, lipoplex particles can be obtained by adding nucleic acid to a colloidal liposome dispersion. Using the ethanol injection technique, such colloidal liposome dispersions are formed, in some embodiments, as follows: an ethanol solution containing lipids, such as cationic or cationic ionizable lipids, and additional lipids is injected into an aqueous solution under stirring.

[0356] The term "particle-forming component" or "particle-forming agent" refers to any component that forms a particle, for example, by associating with a payload. Delivery vehicles, such as particle-forming agents, useful in the present invention include polymers, polymer derivatives, lipids, such as those described herein, and mixtures thereof. Such components include any component that can be part of a nucleic acid particle, for example, cationic or cationic ionizable lipids.

[0357] polymer Polymers are commonly used materials for nanoparticle-based delivery, given their high degree of chemical flexibility. Typically, cationic polymers are used to electrostatically condense negatively charged nucleic acids into nanoparticles. These positively charged groups often consist of amines that change protonation state in the pH range of 5.5 to 7.5, leading to an ionic imbalance that results in endosomal rupture. Polymers such as poly-L-lysine, polyamidoamine, protamine, and polyethyleneimine, as well as naturally occurring polymers such as chitosan, have all been applied to nucleic acid delivery and are suitable as cationic polymers herein. Furthermore, some researchers have synthesized polymers specifically for nucleic acid delivery. Poly(β-amino esters), in particular, are widely used in nucleic acid delivery due to their ease of synthesis and biodegradability. Such synthetic polymers are also suitable as cationic polymers herein.

[0358] As used herein, the term "polymer" is given its usual meaning, i.e., a molecular structure comprising one or more repeating units (monomers) linked by covalent bonds. The repeating units may all be identical, or in some cases, there may be two or more types of repeating units in the polymer. In some cases, the polymer is biologically derived, i.e., a biopolymer such as a protein. In some cases, additional moieties, such as targeting moieties, may also be present in the polymer.

[0359] When two or more types of repeating units are present in a polymer, the polymer is said to be a "copolymer." It should be understood that a polymer as used herein may be a copolymer. The repeating units forming a copolymer may be arranged in any manner. For example, the repeating units may be arranged in a random order, an alternating order, or as a "block" copolymer, i.e., 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). A block copolymer may have two (diblock copolymer), three (triblock copolymer), or more distinct blocks.

[0360] In certain embodiments, the polymer is biocompatible. A biocompatible polymer is typically a polymer that does not cause significant cell death at moderate concentrations. In certain embodiments, the biocompatible polymer is biodegradable, i.e., the polymer can be chemically and / or biologically degraded in a physiological environment, such as within the body.

[0361] In certain embodiments, the polymer may be protamine or a polyalkyleneimine.

[0362] The term "protamine" refers to any of a variety of relatively low molecular weight, strongly basic proteins that are rich in arginine and are found in the sperm cells of various animals (such as fish) in place of somatic histones, particularly in association with DNA. In particular, the term "protamine" refers to a protein found in fish sperm that is strongly basic, soluble in water, does not coagulate with heat, and produces primarily arginine upon hydrolysis. In purified form, they are used to neutralize the anticoagulant effect of heparin in long-acting formulations of insulin.

[0363] In accordance with the present disclosure, the term "protamine" as used herein is intended to include any protamine amino acid sequence and fragments thereof obtained or derived from natural or biological sources, and multimeric forms of said amino acid sequence or fragments thereof, as well as artificial, specifically designed for a particular purpose (synthetic) polypeptides that cannot be isolated from natural or biological sources.

[0364] In some embodiments, the polyalkyleneimine comprises polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine. A preferred polyalkyleneimine is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75×10 2 ~10 7 Da, preferably 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.

[0365] According to the present disclosure, linear polyalkyleneimines such as linear polyethyleneimine (PEI) are preferred.

[0366] Cationic polymers (including polycationic polymers) contemplated for use herein include any cationic polymer that can electrostatically bind to nucleic acids. In some embodiments, cationic polymers contemplated for use herein include any cationic polymer with which nucleic acids can associate, for example, by forming a complex with the nucleic acid or by forming a vesicle in which the nucleic acid is entrapped or encapsulated.

[0367] The particles described herein can also include polymers other than cationic polymers, i.e., non-cationic polymers and / or anionic polymers. Collectively, anionic and neutral polymers are referred to herein as non-cationic polymers.

[0368] Particles containing nucleic acids are also referred to herein as "polyplexes (PLX)". Such PLXs can include lipid components, such as lipid components of targeting compounds. Such particles containing polymers and lipids, such as functionalized lipids, are also referred to as lipidated polyplexes (LPLX).

[0369] Polyamine derivatives (Viromer) In some embodiments, the delivery vehicle comprises a polyamine derivative, e.g., a carboxylated polyamine derivative, which forms polycations in solution that facilitate complexation with polyanions, such as nucleic acids.

[0370] In some embodiments, polyamine derivatives useful in the present invention as delivery vehicles for polyanions include: a polyamine moiety containing multiple amino groups; a plurality of carboxylated substituents comprising carboxyl groups attached to the amino groups of the polyamine moiety via hydrophobic linkers; and a plurality of hydrophobic substituents attached to the amino groups of the polyamine moiety; Includes.

[0371] In some embodiments, polyamine derivatives useful in the present invention as delivery vehicles for polyanions include: a polyamine moiety containing multiple amino groups; a plurality of carboxylated substituents comprising carboxyl groups attached to amino groups of the polyamine moiety via hydrophobic linkers, each of the carboxylated substituents comprising 6 to 40 carbon atoms, preferably 6 to 20 carbon atoms, more preferably 8 to 16 carbon atoms, and each of the hydrophobic linkers may contain 1 to 3 heteroatoms selected from O, N, and S; and a plurality of hydrophobic substituents attached to the amino groups of the polyamine moiety, each of the hydrophobic substituents containing at least 2 carbon atoms, preferably 6 to 40 carbon atoms, and optionally containing 1 to 3 heteroatoms selected from O, N, and S, with the proviso that the hydrophobic substituents have at least 6 carbon atoms.

[0372] In some embodiments, each of the carboxylated substituents of the polyamine derivative comprises any one or more of the following moieties as the hydrophobic linker: alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, arylene, and combinations thereof; and / or Each of the hydrophobic substituents of the polyamine derivatives includes any one or more of the following moieties: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, and combinations thereof.

[0373] In some embodiments, polyamine derivatives useful in the present invention as delivery vehicles for polyanions are polyalkyleneimine derivatives having one or more hydrophobic substituents selected from one or more carboxyalkyl substituents containing 6 to 40 carbon atoms and hydrocarbon substituents having at least 2 carbon atoms, preferably 6 to 40 carbon atoms, each of said hydrophobic substituents may be or include an alkyl group and / or each of said hydrophobic substituents may be or include an aryl group.

[0374] In some embodiments, the polyalkyleneimine is selected from the group consisting of polyethyleneimine, polypropyleneimine, and polybutyleneimine.

[0375] In some embodiments, the polyamine portion of the polyamine derivative may contain 4 to 20,000 nitrogen atoms, more preferably 6 to 10,000 nitrogen atoms, e.g., 6 to 1,000 nitrogen atoms, or 6 to 100 nitrogen atoms per polyamine molecule.

[0376] In some embodiments, the polyamine portion of the polyamine derivative may be a branched polyamine, preferably a branched polyalkyleneimine.

[0377] In some embodiments, the carboxylated substituents contain one or two carboxyl groups, preferably one carboxyl group. In some embodiments, each carboxylated substituent contains 6 to 40 carbon atoms, preferably 6 to 20 carbon atoms, and more preferably 8 to 16 carbon atoms. The hydrophobic linker of the carboxylated substituents can contain one to three, preferably one or two, heteroatoms selected from O, N, and S. Preferably, the heteroatoms are selected from O and S. In one embodiment, one or two heteroatoms selected from O, N, and S, preferably O and S, can be contained in the hydrophobic linker. Thus, the carboxylated substituents can be carboxyhydrocarbyl groups, or they can be carboxyheterohydrocarbyl groups containing one to three heteroatoms selected from O, N, and S, preferably O and S.

[0378] The carboxylated substituents on the polyamine derivative molecule may be exclusively carboxyhydrocarbyl groups, exclusively carboxyheterohydrocarbyl groups, or may be carboxyhydrocarbyl groups and carboxyheterohydrocarbyl groups. In some embodiments, all of the carboxylated substituents are carboxyhydrocarbyl groups. In some embodiments, all of the carboxylated substituents are carboxyheterohydrocarbyl groups.

[0379] When the carboxylated substituent is a carboxyhydrocarbyl group, the hydrocarbyl portion of said carboxyhydrocarbyl group can be a saturated aliphatic hydrocarbyl moiety, an unsaturated aliphatic hydrocarbyl moiety, an alicyclic hydrocarbyl moiety, an aromatic hydrocarbyl moiety, or a moiety comprising two or more moieties from the foregoing list.

[0380] Examples of carboxyhydrocarbyl groups are carboxyalkyl groups, carboxyalkenyl groups, carboxyalkynyl groups, carboxycycloalkyl groups, carboxycycloalkenyl groups, carboxyalkylcycloalkyl groups, carboxycycloalkylalkyl groups, carboxyalkylcycloalkylalkyl groups, carboxyaryl groups, carboxyalkylaryl groups, carboxyarylalkyl groups, and carboxyalkylarylalkyl groups. One, two, or three, preferably one or two, carbon atoms of the hydrocarbyl portion of the carboxylated substituent may be replaced with oxygen, nitrogen, or sulfur, thereby forming a carboxyheterohydrocarbyl moiety. Any such formal replacement with a heteroatom is understood to include adjustment of the attached hydrogen atoms to adjust for the valence of the replaced heteroatom. In preferred embodiments, such carboxyheterohydrocarbyl moieties comprise one or more functional groups in the hydrophobic linker selected from -O-, -S-, -N(H)C(O)-, -C(O)O--OC(O)N(H)-, -C(O)-, -C(O)-N(H)-, -N(H)-C(O)-O-, -OC(O)-, or -SS-.

[0381] In some embodiments, the hydrophobic linker is or includes an alkylene group, such as a linear or branched alkylene group, or the linker is or includes a cycloalkylene group. The alkylene group can be an n-alkylene or isoalkylene group. Examples of alkylene groups include propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, dodecylene, tetradecylene, or hexadecylene groups. Examples of cycloalkylene groups include cyclopentylene, cyclohexylene, and cycloheptylene groups. Examples of alkylcycloalkyl groups include methylcyclopentylene, ethylcyclopentylene, propylcyclopentylene, butylcyclopentylene, pentylcyclopentylene, hexylcyclopentylene, methylcyclohexylene, ethylcyclohexylene, propylcyclohexylene, butylcyclohexylene, pentylcyclohexylene, and hexylcyclohexylene. One or more of these may be combined in a hydrophobic linker.

[0382] In some embodiments, the carboxylated substituent is or includes a carboxyalkyl or carboxycycloalkyl group containing 6 to 20 carbon atoms. Such carboxylated substituents may be selected from the group consisting of carboxy-n-alkyl groups, branched carboxyalkyl groups, or cyclic carboxyalkyl groups and structural or conformational isomers thereof. In preferred embodiments, the carboxyalkyl group is the radical of an acid selected from hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, 2-cyclohexylacetic acid, 4-cyclohexylbutyric acid, 6-cyclohexylhexanoic acid, 2-(2', 3', or 4' ethylcyclohexyl)-acetic acid, 4-(2', 3', or 4' ethylcyclohexyl)-butyric acid, or 6-(2', 3', or 4' ethylcyclohexyl)-hexanoic acid.

[0383] In some embodiments, the hydrophobic linker is or includes an arylene group and has 6 to 20 carbon atoms. The aryl groups forming the arylene group include aromatic hydrocarbyl groups (carbon-only aryl groups) and aromatic heterohydrocarbyl groups (heteroaryl groups). Examples of the former are phenyl, naphthyl, anthracenyl, and phenanthryl. In some embodiments, the nitrogen-containing heteroaryl group has a pK value of <5 to avoid additional positive charges at neutral pH. Examples of such nitrogen-containing heteroaryl groups are indolyl, pyrazinyl, pyridazinyl, pyrimidinyl, cinnolinyl, phthalazinyl, and purinyl. In some embodiments, the oxygen-containing heterohydrocarbyl group forming the hydroxy group has a pK value >12 to avoid negative charges at neutral pH.

[0384] Examples of alkylaryl groups are methylphenyl (tolyl), ethylphenyl, 4-isopropylphenyl, and xylyl groups. Examples of arylalkyl (aralkyl) groups are benzyl, phenylethyl, and trityl groups. Examples of alkylarylalkyl groups are methylbenzyl and 4-isopropylbenzyl groups. The carboxyarylalkyl moiety can be, for example, a radical derived from o-, m-, or p-methylbenzoic acid, or o-, m-, or p-ethylbenzoic acid. The carboxyalkylarylalkyl moiety can be, for example, o-, m-, or p-methylphenylacetic acid. The carboxyalkenylarylalkyl moiety can be, for example, o-, m-, or p-methylcinnamic acid or can be derived therefrom.

[0385] Multiple carboxylated substituents, such as those that are or include carboxyalkyl groups, present on a polyamine derivative can be the same or different. For convenience, they can be the same. The carboxy group of the carboxylated substituent can be attached to any carbon atom of the hydrophobic linker. Preferably, the carboxy group is attached to a carbon atom as follows: if z is the number of carbon atoms in the longest carbon chain in the carboxylated substituent (e.g., carboxyalkyl group) to the carbon atom attached to the polyamine nitrogen atom, and the carbon atom attached to the polyamine nitrogen is counted as position 1, then the carboxy group is attached to the carbon atom at a position that is more than z / 2 atoms away from the polyamine nitrogen. If the value of z / 2 is not an integer, the above definition extends to the position defined by the next integer >z / 2. In one embodiment, the carboxy group is attached to the carbon atom of the hydrophobic linker that is most distal (in terms of the number of carbon atoms) from the polyamine nitrogen atom to which the hydrophobic linker (the alkylene chain in the case of a carboxyalkyl group) is attached. The carboxy group may be attached to the carbon atom in the carboxylated substituent (or carboxyalkyl group) that is farthest from the polyamine nitrogen, for example, the terminal (omega) carbon atom of the carboxylated substituent (or carboxyalkyl group) in the case of a linear carboxylated substituent.

[0386] In some embodiments, the hydrophobic substituent contains 2 to 40 carbon atoms, in some embodiments, 3 to 40 carbon atoms, in some embodiments, 6 to 40 carbon atoms, and in some embodiments, 6 to 20 carbon atoms. The hydrophobic substituent can contain 1 to 3, preferably 1 or 2, heteroatoms selected from O, N, and S, provided that the hydrophobic substituent contains 6 or more carbon atoms. Preferably, the heteroatoms are selected from O and S. Thus, the hydrophobic substituent can be a hydrocarbyl group or a heterohydrocarbyl group, the latter containing 1 to 3 heteroatoms as described above. Among the multiple carboxylated substituents of the polyamine derivative molecule, there can be exclusively hydrocarbyl groups, exclusively heterohydrocarbyl groups, or a mixture of hydrocarbyl and heterohydrocarbyl groups. In some embodiments, all of the multiple hydrophobic substituents are hydrocarbyl groups. In some embodiments, all of the multiple hydrophobic substituents are heterohydrocarbyl groups.

[0387] When the hydrophobic substituent is a hydrocarbyl group, it can be selected from alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, cycloalkenyl groups, cycloalkylalkyl groups, alkylcycloalkyl groups, alkylcycloalkylalkyl groups, aryl groups, alkylaryl groups, arylalkyl groups, and alkylarylalkyl groups, as well as groups containing two or more groups from the above list. When the hydrophobic substituent contains six or more carbon atoms, one, two, or three of the carbon atoms of the hydrocarbyl group can be replaced with oxygen, nitrogen, or sulfur, preferably oxygen or sulfur, thereby forming a heterohydrocarbyl substituent. Such heterohydrocarbyl substituents can contain functional groups selected from -O-, -S-, -N(H)C(O)-, -C(O)O-, -OC(O)N(H)-, -C(O)-, -C(O)-N(H)-, -N(H)-C(O)-O-, -OC(O)-, or -SS-.

[0388] In some embodiments, the hydrophobic substituent is or includes an alkyl group, such as a linear or branched alkyl group, or a cycloalkyl group. The alkyl group can be an n-alkyl or an isoalkyl group. Examples of alkyl groups include propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tetradecyl, or hexadecyl groups. Examples of cycloalkyl groups include cyclopentyl, cyclohexyl, and cycloheptyl groups.

[0389] Examples of alkenyl groups are propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tetradecenyl and hexadecenyl groups. Examples of alkynyl groups are propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl, tetradecynyl and hexadecenyl groups.

[0390] Examples of cycloalkenyl groups are cyclopentenyl, cyclohexenyl, and cycloheptenyl groups. Cycloalkylalkyl groups are groups in which a cycloalkyl group is linked to an alkylene group corresponding to the alkyl group. Examples include cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl, cyclohexylethyl, etc.

[0391] An alkylcycloalkyl group is a group in which an alkyl group is linked to a cycloalkylene group corresponding to the cycloalkyl group. Examples of alkylcycloalkyl groups are methylcyclopentyl, ethylcyclopentyl, propylcyclopentyl, butylcyclopentyl, pentylcyclopentyl, hexylcyclopentyl, methylcyclohexyl, ethylcyclohexyl, propylcyclohexyl, butylcyclohexyl, pentylcyclohexyl, and hexylcyclohexyl.

[0392] An alkylcycloalkylalkyl group is a group in which an alkyl group is linked to a cycloalkylalkylene group.

[0393] In some embodiments, the hydrophobic substituent comprises an aryl group and has 6 to 20, preferably 7 to 15, carbon atoms. Aryl groups include aromatic hydrocarbyl groups (carbon-only aryl groups) and aromatic heterohydrocarbyl groups (heteroaryl groups). Examples of the former are phenyl, naphthyl, and phenanthryl. In some embodiments, the nitrogen-containing heteroaryl group has a pK value of <5 to avoid additional positive charges at neutral pH. Examples of such nitrogen-containing heteroaryl groups are indolyl, pyrazinyl, pyridazinyl, pyrimidinyl, cinnolinyl, phthalazinyl, and purinyl groups. In some embodiments, the oxygen-containing heterohydrocarbyl group forming the hydroxy group has a pK value >12 to avoid negative charges at neutral pH.

[0394] Examples of alkylaryl groups are methylphenyl (tolyl), ethylphenyl, 4-isopropylphenyl, methylindolyl, and xylyl groups. Examples of arylalkyl (aralkyl) groups are benzyl, phenylethyl, indolylmethyl, and trityl groups. Examples of alkylarylalkyl groups are methylbenzyl and 4-isopropylbenzyl groups.

[0395] The different hydrophobic substituents on the molecule of the polyamine derivative can be the same or different. For convenience, they can be the same.

[0396] In some embodiments, the polyamine derivative has a linear polyethyleneimine moiety of 2 to 500 kDa (in terms of number average molecular weight), the carboxylated substituent has 10 to 16 carbon atoms and is an n-alkylcarboxylic acid, and the hydrophobic substituent has 1 to 12 carbon atoms and is alkyl, preferably n-alkyl, and / or alkylarylalkyl.

[0397] In some embodiments, the polyamine derivative has a branched polyethyleneimine moiety of 0.5 to 200 kDa (in terms of number average molecular weight), the carboxylated substituent has 10 to 16 carbon atoms and is an n-alkylcarboxylic acid, and the hydrophobic substituent has 1 to 12 carbon atoms and is alkyl, preferably n-alkyl, and / or alkylarylalkyl.

[0398] lipids The terms "lipid" and "lipid-like substance" are broadly defined herein as molecules containing one or more hydrophobic moieties or groups and, optionally, one or more hydrophilic moieties or groups. Molecules containing both hydrophobic and hydrophilic moieties are often referred to as amphiphiles. Lipids are typically insoluble or poorly soluble in water, but are soluble in many organic solvents. In aqueous environments, their amphiphilic nature allows them to self-assemble into organized structures and various phases. One of these phases consists of lipid bilayers when they exist in aqueous environments as vesicles, multilamellar / unilamellar liposomes, or membranes. Hydrophobicity can be imparted by the inclusion of nonpolar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Hydrophilic groups can include polar and / or charged groups, including carbohydrates, phosphate groups, carboxylate groups, sulfate groups, amino groups, sulfhydryl groups, nitro groups, hydroxyl groups, and other similar groups.

[0399] As used herein, the term "hydrophobic" refers to any molecule, moiety, or group that is substantially immiscible or insoluble in aqueous solution. The term hydrophobic group includes hydrocarbons having at least six carbon atoms. Monovalent radicals of hydrocarbons are referred to herein as hydrocarbyls. Hydrophobic groups can have functional groups (e.g., ethers, esters, halides, etc.) and atoms other than carbon and hydrogen, so long as they are substantially immiscible or insoluble in aqueous solution.

[0400] The term "hydrocarbon" includes acyclic, e.g., straight-chain (linear) or branched-chain hydrocarbyl groups, such as alkyl, alkenyl, or alkynyl, as defined herein. It is understood that one or more of the hydrogens in an alkyl, alkenyl, or alkynyl may be replaced with other atoms, such as halogen, oxygen, or sulfur. Unless otherwise specified, hydrocarbon groups can also include cyclic (alkyl, alkenyl, or alkynyl) groups or aryl groups, so long as the overall polarity of the hydrocarbon remains relatively nonpolar.

[0401] The term "alkyl" refers to a saturated straight- or branched-chain monovalent hydrocarbon moiety which can have 1 to 30, typically 1 to 20, and often 6 to 18 carbon atoms. Exemplary nonpolar alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, hexyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, and the like.

[0402] The term "alkenyl" refers to a straight- or branched-chain monovalent hydrocarbon moiety having at least one carbon-carbon double bond, which may contain 2 to 30 total carbon atoms, typically 6 to 20, and often 6 to 18 carbon atoms. Generally, the maximum number of carbon-carbon double bonds in an alkenyl group can be equal to the integer calculated by dividing the number of carbon atoms in the alkenyl group by 2; if the number of carbon atoms in the alkenyl group is odd, the result of the division is rounded down to the next 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), i.e., 1, 2, 3, 4, 5, or 6 carbon-carbon double bonds.

[0403] The term "alkynyl" refers to a straight- or branched-chain monovalent hydrocarbon moiety having at least one carbon-carbon triple bond and a total of 2 to 30, typically 6 to 20, and often 6 to 18 carbon atoms. An alkynyl group may have one or more carbon-carbon double bonds. Generally, the maximum number of carbon-carbon triple bonds in an alkynyl group can be equal to the integer calculated by dividing the number of carbon atoms in the alkynyl group by 2; if the number of carbon atoms in the alkynyl group is odd, the result of the division is rounded down to the next 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 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, more preferably 1 or 2, carbon-carbon triple bonds.

[0404] The term "alkylene" refers to a saturated straight- or branched-chain divalent hydrocarbon moiety which can have 1 to 30, typically 2 to 20, and often 4 to 12 carbon atoms. Exemplary nonpolar alkylene groups include, but are not limited to, methylene, ethylene, trimethylene, hexamethylene, decamethylene, dodecamethylene, tetradecamethylene, hexadecamethylene, octademethylene, and the like.

[0405] The term "alkenylene" refers to a straight- or branched-chain divalent hydrocarbon moiety having at least one carbon-carbon double bond and a total of 2 to 30, typically 2 to 20, and often 4 to 12 carbon atoms. Generally, the maximum number of carbon-carbon double bonds in an alkenylene group can be equal to the integer calculated by dividing the number of carbon atoms in the alkenylene group by 2; if the number of carbon atoms in the alkenylene group is odd, the result of the division is rounded down to the next integer. For example, for an alkenylene group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenylene group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5, or 6 carbon-carbon double bonds.

[0406] The term "alkenylene" refers to a straight- or branched-chain divalent hydrocarbon moiety having at least one carbon-carbon triple bond and which may contain a total of 2 to 30 carbon atoms, typically 2 to 20, and often 4 to 12. An alkynyl group may have one or more carbon-carbon double bonds.

[0407] The term "cycloalkyl" refers to cyclic, non-aromatic versions of "alkyl" and "alkenyl," preferably having 3 to 14 carbon atoms, e.g., 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 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cyclodecyl, cyclodecenyl, and adamantyl. Cycloalkyl groups can consist of one ring (monocyclic), two rings (bicyclic), or three or more rings (polycyclic).

[0408] The term "aryl" refers to a monoradical of an aromatic cyclic hydrocarbon. Preferably, an aryl group contains 3 to 14 (e.g., 5, 6, 7, 8, 9, or 10, e.g., 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 cyclopropenylium, cyclopentadienyl, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, and phenanthryl. Preferably, "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. Aryl does not include fullerenes.

[0409] The term "aromatic" used in the context of hydrocarbons means that the entire molecule must be aromatic. For example, if a monocyclic aryl is hydrogenated (partially or fully), the resulting hydrogenated ring structure is classified as a cycloalkyl for purposes of this disclosure. Similarly, if a bicyclic or polycyclic aryl (such as naphthyl) is hydrogenated, the resulting hydrogenated bicyclic or polycyclic structure (such as 1,2-dihydronaphthyl) is classified as a cycloalkyl for purposes of this disclosure (even if one ring is still aromatic, as in 1,2-dihydronaphthyl).

[0410] As used herein, the term "amphiphilic" refers to a molecule having both polar and non-polar portions. Amphiphilic compounds often have a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non-polar portion is insoluble in water. Furthermore, the polar portion may have either a formal positive or a formal negative charge. Alternatively, the polar portion may have both a formal positive and a formal negative charge, or may be a zwitterion or an internal salt. For purposes of this disclosure, an amphiphilic compound may be, but is not limited to, one or more natural or non-natural lipids and lipid-like compounds.

[0411] The terms "lipid-like substance," "lipid-like compound," or "lipid-like molecule" refer to substances, particularly amphiphiles, that are structurally and / or functionally related to lipids but cannot be considered lipids in the strict sense. For example, the term includes compounds that can form amphiphilic layers when present in vesicles, multilamellar / unilamellar liposomes, or membranes in aqueous environments, as well as surfactants or synthetic compounds with both hydrophilic and hydrophobic moieties. Generally speaking, the term includes molecules containing hydrophilic and hydrophobic moieties with different structural organizations that may or may not resemble those of lipids. Examples of lipid-like compounds capable of spontaneous integration into cell membranes include functional lipid constructs such as synthetic function-spacer-lipid constructs (FSLs), synthetic function-spacer-sterol constructs (FSSs), and artificial amphiphilic molecules. Lipids containing two long alkyl chains and a polar head group are generally cylindrical. The area occupied by the two alkyl chains is similar to the area occupied by the polar head group. Such lipids have low solubility as monomers and tend to aggregate into water-insoluble planar bilayers. Conventional surfactant monomers, containing only a single linear alkyl chain and a hydrophilic head group, are generally conical in shape. The hydrophilic head group tends to occupy more molecular space than the linear alkyl chain. In some embodiments, surfactants tend to aggregate into spherical or ellipsoidal micelles that are water-soluble. Lipids also have the same general structure as surfactants—a polar hydrophilic head group and a nonpolar hydrophobic tail—but lipids differ from surfactants in the shape of the monomer, the type of aggregates they form in solution, and the concentration range required for aggregation. As used herein, the term "lipid" should be interpreted to encompass both lipids and lipid-like substances, unless otherwise indicated herein or clearly contradicted by context.

[0412] Generally, lipids can be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from the condensation of ketoacyl subunits), sterol lipids, and prenol lipids (derived from the condensation of isoprene subunits). The term "lipid" is sometimes used synonymously with fat, but fat is a subgroup of lipids called triglycerides. Lipids also include molecules such as fatty acids and their derivatives (including triglycerides, diglycerides, monoglycerides, and phospholipids), as well as sterol-containing metabolites such as steroids, i.e., cholesterol or its derivatives. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and their derivatives, and mixtures thereof.

[0413] Fatty acids or fatty acid residues are a diverse group of molecules consisting of a hydrocarbon chain terminated in a carboxylic acid group; this arrangement gives the molecule a polar, hydrophilic end and a nonpolar, hydrophobic end that is insoluble in water. The carbon chain, typically 4 to 24 carbons long, can be saturated or unsaturated and may be attached to functional groups including oxygen, halogens, nitrogen, and sulfur. When fatty acids contain double bonds, they can be either cis or trans geometric isomers, which significantly affect the molecular configuration. Cis double bonds cause the fatty acid chain to bend, an effect that, combined with more double bonds in the chain, can have a significant effect. Other major lipid classes in the fatty acid category are fatty acid esters and fatty acid amides.

[0414] Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the most well-known of which are fatty acid triesters of glycerol called triglycerides. The term "triacylglycerol" is sometimes used synonymously with "triglyceride." In these compounds, each of the three hydroxyl groups of glycerol is typically esterified with a different fatty acid. A further subclass of glycerolipids is represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via glycosidic bonds.

[0415] Glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core attached by ester bonds to two fatty acid-derived "tails" and by a phosphate ester bond to a "head" group. Examples of glycerophospholipids, commonly called phospholipids (although sphingomyelin is also classified as a phospholipid), are phosphatidylcholine (PC, also known as GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn), and phosphatidylserine (PS or GPSer).

[0416] Sphingolipids are a complex family of compounds that share a common structural feature: a sphingoid base backbone. The predominant sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with amide-linked fatty acids. The fatty acids are typically saturated or monounsaturated, with chain lengths of 16–26 carbon atoms. The predominant sphingophospholipid in mammals is sphingomyelin (ceramide phosphocholine), while insects contain primarily ceramide phosphoethanolamine, and fungi have phytoceramide phosphoinositol and mannose-containing head groups. Glycosphingolipids are a diverse family of molecules composed of one or more sugar residues attached to a sphingoid base via glycosidic bonds. Examples of these are simple and complex glycosphingolipids such as cerebrosides and gangliosides.

[0417] Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, are important components of membrane lipids, along with glycerophospholipids and sphingomyelins.

[0418] Saccharolipids refer to compounds in which fatty acids are directly linked to a sugar backbone, forming structures compatible with membrane bilayers. In saccharolipids, monosaccharides replace the glycerol backbone present in glycerolipids and glycerophospholipids. The best-known saccharolipid is the acylated glucosamine precursor of the lipid A component of the lipopolysaccharide of Gram-negative bacteria. A typical lipid A molecule is a disaccharide of glucosamine derivatized with as many as seven fatty acyl chains. The minimal lipopolysaccharide required for growth in Escherichia coli is Kdo2-lipid A, a hexaacylated disaccharide of glucosamine glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.

[0419] Polyketides are synthesized by the polymerization of acetyl and propionyl subunits by classical enzymes, as well as by iterative and multimodular enzymes that share mechanistic features with fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal, and marine sources, and possess great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

[0420] According to the present disclosure, lipids and lipid-like substances can be cationic, anionic, or neutral. Neutral lipids or lipid-like substances exist in an uncharged or neutral zwitterionic form at a selected pH.

[0421] Cationic / cationic ionizable lipids In some embodiments, the particles described herein comprise at least one cationic or cationic ionizable lipid as particle-forming agent.The cationic or cationic ionizable lipid contemplated for use herein comprises any cationic or cationic ionizable lipid (including lipid-like substances) that can electrostatically bind to nucleic acid.In some embodiments, the cationic or cationic ionizable lipid contemplated for use herein can be associated with nucleic acid, for example, by forming a complex with nucleic acid or by forming vesicles in which nucleic acid is enclosed or encapsulated.

[0422] As used herein, "cationic lipid" refers to a lipid or lipid-like substance that has a net positive charge. Generally, cationic lipids have a lipophilic moiety such as a sterol, an acyl chain, a diacyl or higher acyl chain, and the head group of the lipid typically carries the positive charge.

[0423] In some embodiments, cationic lipid only has net positive charge at certain pH, particularly acidic pH, but at different, preferably higher pH such as physiological pH, preferably does not have net positive charge, preferably has no charge, i.e., is neutral.This ionizable behavior is thought to improve efficacy by helping endosomal escape and reducing toxicity, compared with particles that remain cationic at physiological pH.

[0424] As used herein, " cationic ionizable lipid " refers to the lipid or lipid-like substance that has net positive charge or is neutral, that is, is not permanently cationic.Therefore, depending on the pH of the composition in which cationic ionizable lipid is dissolved, cationic ionizable lipid is either positively charged or neutral.For the purpose of the present disclosure, cationic ionizable lipid is included in the term " cationic lipid " unless otherwise contradicted by the context.

[0425] In some embodiments, cationic or cationic-ionizable lipids comprise a head group that includes at least one nitrogen atom (N) that is positively charged or capable of being protonated, e.g., under physiological conditions.

[0426] Examples of cationic or cationic ionizable lipids include N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 3-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammoniumpropane (DODAP); 1,2-Diacyloxy-3-dimethylammonium propane; 1,2-Dialkyloxy-3-dimethylammonium propane; Dioctadecyldimethylammonium chloride (DODAC), 1,2-Distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-Di(tetradecoxy)propyl-(2-hydroxyethyl)dimethylazanium (DMRIE), 1,2-Dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-Dimyristoyl-3-trimethylammonium propane (DM TAP), 1,2-dioleyloxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycylspermine (DOGS), 3-dimethylamino-2- (Cholest-5-ene-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienooxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienooxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-D MA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9- (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecenyloxy)-1-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide nium bromide (βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium propane (DMDAP), 1,2-Dipalmitoyl-3-dimethylammonium propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropan-1-aminium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide (DMORIE), di((Z)-non-2-en-1-yl)8,8'-((((2 (Dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)ethyl]-amino}-ethylamino)propionamide (Lipidoid 98N, 12 -5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (Lipidoid C12-200).

[0427] In some embodiments, the cationic or cationic ionizable lipid is DOTMA. In some embodiments, the cationic or cationic ionizable lipid is DODMA.

[0428] DOTMA is a cationic lipid with a quaternary amine head group. The structure of DOTMA can be represented as follows: [ka]

[0429] DODMA is an ionizable cationic lipid with a tertiary amine head group. The structure of DODMA can be represented as follows: [ka]

[0430] In some embodiments, the cationic or cationic ionizable lipid may comprise from about 10 mol% to about 95 mol%, from about 20 mol% to about 95 mol%, from about 20 mol% to about 90 mol%, from about 30 mol% to about 90 mol%, from about 40 mol% to about 90 mol%, or from about 40 mol% to about 80 mol% of the total lipid present in the particle.

[0431] More lipids The particles described herein can also comprise lipids (including lipid-like substances) other than cationic or cationic ionizable lipids (collectively referred to herein as cationic lipids), i.e., non-cationic lipids (including non-cationic or non-cationic ionizable lipids or lipid-like substances).Collectively, anionic and neutral lipids or lipid-like substances are referred to herein as non-cationic lipids.In addition to cationic or cationic ionizable lipids, the formulation of nucleic acid particles can be optimized by adding other hydrophobic moieties such as cholesterol and lipids to improve particle stability and the effectiveness of nucleic acid delivery.

[0432] One or more additional lipids may or may not affect the overall charge of nucleic acid particles.In some embodiments, one or more additional lipids are non-cationic lipids or lipid-like substances.Non-cationic lipids may include, for example, one or more anionic lipids and / or neutral lipids.As used herein, "anionic lipid" refers to any lipid that is negatively charged at a selected pH.As used herein, "neutral lipid" refers to any of several lipid species that exist in uncharged or neutral zwitterionic form at a selected pH.

[0433] In some embodiments, the nucleic acid particles described herein comprise a cationic or cationic ionizable lipid and one or more additional lipids.

[0434] Without wishing to be bound by theory, the amount of cationic or cationic ionizable lipid relative to the amount of one or more additional lipids can affect important nucleic acid particle properties, such as nucleic acid charge, particle size, stability, tissue selectivity, and biological activity. Thus, in some embodiments, the molar ratio of cationic or cationic ionizable lipid to one or more additional lipids is about 10:0 to about 1:9, about 4:1 to about 1:2, about 4:1 to about 1:1, about 3:1 to about 1:1, or about 3:1 to about 2:1.

[0435] In some embodiments, the one or more additional lipids included in the nucleic acid particles described herein include one or more of a neutral lipid, a steroid, and combinations thereof.

[0436] In some embodiments, one or more additional lipids comprise the neutral lipid that is a phospholipid.In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine and sphingomyelin.The specific phospholipid that can be used includes but is not limited to phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine or sphingomyelin.Such phospholipids include, in particular, diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBP), diacylphosphatidylcholine (DPC ... PC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC) and phosphatidylethanolamines, in particular diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (D PyPE), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM), and additional phosphatidylethanolamine lipids with different hydrophobic chains. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM.In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC. In some embodiments, the neutral lipid is DOPE.

[0437] In some embodiments, the additional lipid comprises one of the following: (1) a phospholipid, (2) cholesterol or a derivative thereof, or (3) a mixture of a phospholipid and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.

[0438] Thus, in some embodiments, the nucleic acid particles described herein comprise (1) a cationic or cationic ionizable lipid and a phospholipid such as DSPC or DOPE, or (2) a cationic or cationic ionizable lipid and a phospholipid such as DSPC or DOPE and cholesterol.

[0439] In some embodiments, the nucleic acid particles described herein comprise (1) DOTMA and DOPE, (2) DOTMA, DOPE and cholesterol, (3) DODMA and DOPE, or (4) DODMA, DOPE and cholesterol.

[0440] DSPC is a neutral phospholipid. The structure of DSPC can be represented as follows: [ka]

[0441] DOPE is a neutral phospholipid. The structure of DOPE can be represented as follows: [ka]

[0442] The structure of cholesterol can be represented as follows: [ka]

[0443] In some embodiments, the nucleic acid particles described herein do not include polymer-conjugated lipids, such as PEGylated lipids.

[0444] In some embodiments, the additional lipids (e.g., one or more phospholipids and / or cholesterol) may comprise about 0 mol% to about 90 mol%, about 0 mol% to about 80 mol%, about 2 mol% to about 80 mol%, about 5 mol% to about 80 mol%, about 5 mol% to about 60 mol%, about 5 mol% to about 50 mol%, about 7.5 mol% to about 50 mol%, or about 10 mol% to about 40 mol% of the total lipid present in the particle. In some embodiments, the additional lipids (e.g., one or more phospholipids and / or cholesterol) comprise about 10 mol%, about 15 mol%, or about 20 mol% of the total lipid present in the particle.

[0445] In some embodiments, the additional lipid comprises a mixture of (i) a phospholipid, such as DOPE, and (ii) cholesterol or a derivative thereof, in which the molar ratio of the phospholipid, such as DOPE, to cholesterol or a derivative thereof is about 9:0 to about 1:10, about 2:1 to about 1:4, about 1:1 to about 1:4, or about 1:1 to about 1:3.

[0446] Polymer-bound lipids In some embodiments, the particles described herein may include at least one polymer-bound lipid. In some embodiments, the polymer-bound lipid includes an amphiphilic derivative of a polymer that is part of the targeting compound and / or a polymer-bound lipid that is not part of the targeting compound. The polymer-bound lipid is typically a molecule comprising a lipid portion and a polymer portion attached thereto. In some embodiments, the polymer of the polymer-bound lipid is a polymer described herein for the targeting compound. In some embodiments, the polymer-bound lipid is a PEG-bound lipid, also referred to herein as a PEGylated lipid or PEG lipid. The term "PEGylated lipid" refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art. In some embodiments, the polymer-bound lipid is a polysarcosine-bound lipid, also referred to herein as a sarcosinylated lipid or pSar lipid. The term "sarcosinylated lipid" refers to a molecule comprising both a lipid portion and a polysarcosine portion.

[0447] In some embodiments, the polymer-bound lipid is designed to sterically stabilize the lipid particle by forming a protective hydrophilic layer that shields the hydrophobic lipid layer. In some embodiments, the polymer-bound lipid can reduce its association with serum proteins and / or the resulting uptake by the reticuloendothelial system when such lipid particles are administered in vivo.

[0448] Polyethylene glycol (PEG)-conjugated lipids In some embodiments, the particles described herein comprise a PEG-linked lipid.

[0449] In some embodiments, the PEG-conjugated lipid (PEGylated lipid) has the following general formula: [ka] (In the formula, R 12 and R 13each independently represents a straight or branched alkyl or alkenyl chain containing 10 to 30 carbon atoms, wherein the alkyl / alkenyl chain may be interrupted by one or more ester bonds, and w has an average value in the range of 30 to 60. or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0450] In some embodiments of this formula, R 12 and R 13 are each independently a straight alkyl chain containing 10 to 18 carbon atoms, preferably 12 to 16 carbon atoms.

[0451] In some embodiments of this formula, R 12 and R 13 are identical. In some embodiments, R 12 and R 13 Each of R is a linear alkyl chain containing 12 carbon atoms. 12 and R 13 Each of R is a linear alkyl chain containing 14 carbon atoms. 12 and R 13 Each of the is a straight alkyl chain containing 16 carbon atoms.

[0452] In some embodiments of this formula, R 12 and R 13 In some embodiments, R 12 and R 13 is a linear alkyl chain containing 12 carbon atoms, and R 12 and R 13 The other is a straight alkyl chain containing 14 carbon atoms.

[0453] In some embodiments of this formula, w has an average value in the range of 40-50, for example, an average value of 45.

[0454] In some embodiments of this formula, w is within a range such that the PEG portion of the PEGylated lipid has an average molecular weight of about 400 to about 6000 g / mol, e.g., about 1000 to about 5000 g / mol, about 1500 to about 4000 g / mol, or about 2000 to about 3000 g / mol. 12 and R 13 Each of is a linear alkyl chain containing 14 carbon atoms, and w has an average value of 45.

[0455] Various PEG-conjugated lipids are known in the art, including, but not limited to, PEGylated diacylglycerols (PEG-DAGs), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG diacylglycerol succinate (PEG-S-DAGs), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), PEGylated ceramide (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.

[0456] In some embodiments, the PEG-conjugated lipid (PEGylated lipid) is or comprises 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide. In some embodiments, the PEGylated lipid has the following structure: [ka]

[0457] In some embodiments, the PEG-conjugated lipid (PEGylated lipid) is, for example, DMG-PEG2000, having the following structure: [ka]

[0458] In some embodiments, the PEG-conjugated lipid (PEGylated lipid) has the following structure: (wherein n is in the range of 30 to 60, for example, has an average value of about 50) In some embodiments, [ka] PEG-conjugated lipids (PEGylated lipids) preferably refer to 3-N-[(ω-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxy-propylamine (MPEG-(2kDa)-C-DMA) or methoxy-polyethylene glycol-2,3-bis(tetradecyloxy)propylcarbamate (2000), PEG 2000 -C-DMA.

[0459] In some embodiments, the nucleic acid particles described herein may include one or more PEG-conjugated or PEGylated lipids, such as those described in WO 2017 / 075531 and WO 2018 / 081480, the entire contents of each of which are incorporated herein by reference for the purposes described herein.

[0460] In some embodiments, PEGylated lipids comprise from about 1 mol % to about 10 mol %, preferably from about 1 mol % to about 5 mol %, and more preferably from about 1 mol % to about 2.5 mol % of the total lipids present in the nucleic acid compositions / formulations and nucleic acid particles described herein.

[0461] Lipoplex Particle Embodiments In some embodiments of the present disclosure, nucleic acids such as RNA described herein may be present in lipoplex particles.

[0462] Lipoplexes (LPXs) are generally electrostatic complexes formed by mixing preformed cationic lipid liposomes with anionic nucleic acids. The resulting lipoplexes have distinct internal molecular arrangements resulting from the conversion of the liposomal structure into compact nucleic acid lipoplexes.

[0463] In some embodiments, liposomes are self-closed unilamellar or multilamellar vesicular particles, the lamellae of which comprise a lipid bilayer and the enclosed lumen of which comprises an aqueous phase. A prerequisite for using liposomes to form nanoparticles is that they can form a lamellar (bilayer) phase in an aqueous environment to which the lipids in the mixture are applied as needed.

[0464] In certain embodiments, the nucleic acid lipoplex particles comprise both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

[0465] In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In certain embodiments, the molar ratio can be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2:1.

[0466] The nucleic acid lipoplex particles described herein, in some embodiments, have an average diameter in the range of about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 250 nm to about 700 nm, about 400 nm to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm. In certain embodiments, the RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 675 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm. In some embodiments, the nucleic acid lipoplex particles have an average diameter in the range of about 250 nm to about 700 nm. In some embodiments, the RNA lipoplex particles have an average diameter ranging from about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.

[0467] In some embodiments, the functionalized particles described herein are lipid particles in specific lipoplexes containing an RNA drug substance, 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000-alfa peptide (DSPE-PEG2k-alfa), and tetradecyl-poly(sarcosine) 23-acetate (C14-PSar(23)-Ac). The functionalized lipoplexes are produced using a two-step protocol: (1) production of alpha-tagged RNA particles, followed by (2) functionalization of the alpha-tagged lipoplexes with a ligand. The alpha-tagged RNA-lipid particles can be prepared using aqueous / aqueous or aqueous / organic production protocols.

[0468] Lipid Nanoparticle (LNP) Embodiments In some embodiments, the nucleic acids described herein are present in the form of lipid nanoparticles (LNPs). LNPs can include any lipid capable of forming particles to which one or more nucleic acid molecules are bound or encapsulated.

[0469] Generally, lipid nanoparticles can be obtained by directly mixing nucleic acids, such as RNA, in an aqueous phase with lipids in a phase containing an organic solvent, such as ethanol. In this case, lipids or lipid mixtures that do not form a lamellar (bilayer) phase in water can be used for particle formation.

[0470] LNPs typically contain four components: a cationic ionizable lipid, a neutral lipid such as a phospholipid, a steroid such as cholesterol, and a polymer-conjugated lipid such as a PEG-lipid. LNPs can be prepared by mixing lipids dissolved in ethanol with nucleic acid in an aqueous buffer.

[0471] In some embodiments, the LNP comprises 40-60 mol%, 40-55 mol%, 45-55 mol%, or 45-50 mol% cationic ionizable lipids.

[0472] In some embodiments, the neutral lipid is present at a concentration ranging from 5 to 15 mol%, 7 to 13 mol%, or 9 to 11 mol%.

[0473] In some embodiments, the steroid is present at a concentration ranging from 30-50 mol%, 30-45 mol%, 35-45 mol%, or 35-43 mol%.

[0474] In some embodiments, the LNP comprises 1-10 mol%, 1-5 mol%, or 1-2.5 mol% polymer-conjugated lipid.

[0475] In some embodiments, the LNPs comprise 45-55 mol% cationic ionizable lipids; 5-15 mol% neutral lipids; 30-45 mol% steroids; 1-5 mol% polymer-conjugated lipids; and nucleic acids encapsulated within or associated with the lipid nanoparticles.

[0476] In some embodiments, the mole percent is determined based on the total moles of lipid present in the lipid nanoparticle, hi some embodiments, the mole percent is determined based on the total moles of cationic ionizable lipid, neutral lipid, steroid, and polymer-bound lipid present in the lipid nanoparticle.

[0477] In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the neutral lipid is DSPC.

[0478] In some embodiments, the steroid is cholesterol.

[0479] In some embodiments, the polymer-bound lipid is a pegylated lipid, such as the pegylated lipids described above.

[0480] In some embodiments, the cationic ionizable lipid component of the LNP has the structure of formula (III): [ka] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: L 1 or L 2 One of the following is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NRa C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -or-NR a C(=O)O-, and L 1 or L 2 The other is -O(C=O)-, -(C=O)O-, -C(=O)O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -or-NR a C(=O)O- or a direct bond; G 1 and G 2 are each independently an unsubstituted C-C 12 Alkylene or C1-C 12 alkenylene; G 3 is C1-C 24 Alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; R a is H or C1-C 12 is alkyl; R 1 and R 2 are independently C6-C 24 Alkyl or C6-C 24 is alkenyl; R 3 H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 and; R 4 is C1-C 12 is alkyl; R 5 is H or C1-C6 alkyl; and x is 0, 1 or 2.

[0481] In some of the foregoing embodiments of formula (III), the lipid has the following structure (IIIA) or (IIIB): [ka] and where: A is a 3-8 membered cycloalkyl or cycloalkylene ring; R 6 is, in each occurrence, independently H, OH, or C-C 24 is alkyl; n is an integer ranging from 1 to 15.

[0482] In some of the foregoing embodiments of formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[0483] In other embodiments of formula (III), the lipid has the following structure (IIIC) or (IIID): [ka] wherein y and z are each independently an integer in the range of 1 to 12.

[0484] In any of the foregoing embodiments of Formula (III), L 1 or L 2 One of L is -O(C=O)-. For example, in some embodiments, L 1 and L 2 Each of L is -O(C=O)-. In some different embodiments of any of the foregoing, L 1 and L 2 are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments, L 1 and L 2 Each of is —(C═O)O—.

[0485] In some different embodiments of formula (III), the lipid has the following structure (IIIE) or (IIIF): [ka] It has one of the following.

[0486] In some of the foregoing embodiments of formula (III), the lipid has the following structure (IIIG), (IIIH), (IIII) or (IIIJ): [ka] It has one of the following.

[0487] In some of the foregoing embodiments of Formula (III), n is an integer ranging from 2 to 12, e.g., from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5, or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0488] In some other embodiments of the foregoing embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.

[0489] In some of the foregoing embodiments of formula (III), R 6 is H. In other embodiments of the foregoing embodiments, R 6 is C1-C 24 In another embodiment, R 6 is OH.

[0490] In some embodiments of Formula (III), G 3 is unsubstituted. In other embodiments, G is substituted. In various different embodiments, G 3 is a linear C1-C 24 Alkylene or straight chain C1-C 24It is alkenylene.

[0491] In some other aforementioned embodiments of formula (III), R 1 Or R 2 , or both C6-C 24 For example, in some embodiments, R 1 and R 2 are each independently of the following structure: [ka] where R 7a and R 7b is, in each occurrence, independently H or C1-C 12 is alkyl; and a is an integer from 2 to 12, where R 7a , R 7b and a are R 1 and R 2 are each independently selected to contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer ranging from 5 to 9 or 8 to 12.

[0492] In some of the foregoing embodiments of formula (III), R 7a At least one occurrence of is H. For example, in some embodiments, R 7a is H at each occurrence. In other variations of the foregoing embodiments, R 7b is C1-C8 alkyl. For example, in some embodiments, the C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0493] In different embodiments of formula (III), R 1 Or R 2 , or both, have the following structure: [ka] It has one of the following.

[0494] In some of the foregoing embodiments of formula (III), R 3 OH, CN, -C(=O)OR 4 , -OC(=O)R 4 or -NHC(=O)R 4 In some embodiments, R 4 is methyl or ethyl.

[0495] In various different embodiments, the cationic lipid of formula (III) has one of the structures shown in the table below. Representative compounds of formula (III). [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0496] Further exemplary cationic ionizable lipids are: [ka]

[0497] In some embodiments, nucleic acids, such as RNA, described herein are formulated in LNP compositions comprising cationic ionizable lipids, e.g., the cationic ionizable lipids set forth above, neutral lipids, steroids, and polymer-conjugated lipids.

[0498] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising a cationic ionizable lipid of formula III, a neutral lipid, a steroid, and a polymer-conjugated lipid.

[0499] In some embodiments, nucleic acids, such as RNA, described herein are formulated in LNP compositions comprising cationic ionizable lipids, neutral lipids, steroids, and polymer-bound lipids as shown in the table above.

[0500] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising 3D-P-DMA, a neutral lipid, a steroid, and a polymer-conjugated lipid.

[0501] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising ALC-0366, a neutral lipid, a steroid, and a polymer-conjugated lipid.

[0502] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising ALC-0315, a neutral lipid, a steroid, and a polymer-conjugated lipid.

[0503] In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the polymer-bound lipid is a PEGylated lipid, e.g., DMG-PEG2000, PEG 2000 -C-DMA, or ALC-0159.

[0504] In some embodiments, nucleic acids, such as RNA, described herein are formulated in LNP compositions comprising cationic ionizable lipids, e.g., the cationic ionizable lipids set forth above, neutral lipids, steroids, and PEGylated lipids.

[0505] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising a cationic ionizable lipid of formula III, a neutral lipid, a steroid, and a PEGylated lipid.

[0506] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising a cationic ionizable lipid, a neutral lipid, a steroid, and a PEGylated lipid as shown in the table above.

[0507] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising 3D-P-DMA, a neutral lipid, a steroid, and a PEGylated lipid.

[0508] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising ALC-0366, a neutral lipid, a steroid, and a PEGylated lipid.

[0509] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising ALC-0315, a neutral lipid, a steroid, and a PEGylated lipid.

[0510] In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the pegylated lipid is DMG-PEG2000, PEG 2000 -C-DMA, or ALC-0159.

[0511] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising a cationic ionizable lipid, e.g., a cationic ionizable lipid set forth above, DSPC, cholesterol, and a PEGylated lipid.

[0512] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising a cationic ionizable lipid of formula III, DSPC, cholesterol, and a PEGylated lipid.

[0513] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising a cationic ionizable lipid, DSPC, cholesterol, and a PEGylated lipid as shown in the table above.

[0514] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and a PEGylated lipid.

[0515] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising ALC-0366, DSPC, cholesterol, and a PEGylated lipid.

[0516] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising ALC-0315, DSPC, cholesterol, and a PEGylated lipid.

[0517] In some embodiments, the PEGylated lipid is DMG-PEG2000, PEG 2000 -C-DMA, or ALC-0159.

[0518] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising a cationic ionizable lipid, e.g., a cationic ionizable lipid as set forth above, DSPC, cholesterol, and DMG-PEG2000.

[0519] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising a cationic ionizable lipid of formula III, DSPC, cholesterol, and DMG-PEG2000.

[0520] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising a cationic ionizable lipid, DSPC, cholesterol, and DMG-PEG2000 as shown in the table above.

[0521] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and DMG-PEG2000.

[0522] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising ALC-0366, DSPC, cholesterol, and DMG-PEG2000.

[0523] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising ALC-0315, DSPC, cholesterol, and DMG-PEG2000.

[0524] In some embodiments, nucleic acids, such as RNA, described herein, are soluble in cationic ionizable lipids, such as those set forth above, DSPC, cholesterol, and PEG. 2000 -Formulated in an LNP composition containing C-DMA.

[0525] In some embodiments, a nucleic acid, such as an RNA, described herein is prepared by cleaving a nucleic acid molecule containing a cationic ionizable lipid of formula III, DSPC, cholesterol, and PEG. 2000 -Formulated in an LNP composition containing C-DMA.

[0526] In some embodiments, nucleic acids, such as RNA, described herein are synthesized using cationic ionizable lipids, DSPC, cholesterol, and PEG as shown in the table above. 2000 -Formulated in an LNP composition containing C-DMA.

[0527] In some embodiments, nucleic acids such as RNA described herein are prepared using 3D-P-DMA, DSPC, cholesterol, and PEG. 2000 -Formulated in an LNP composition containing C-DMA.

[0528] In some embodiments, the nucleic acids, such as RNA described herein, are synthesized using ALC-0366, DSPC, cholesterol, and PEG. 2000 -Formulated in an LNP composition containing C-DMA.

[0529] In some embodiments, the nucleic acids, such as RNA described herein, are synthesized using ALC-0315, DSPC, cholesterol, and PEG. 2000 -Formulated in an LNP composition containing C-DMA.

[0530] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising a cationic ionizable lipid, e.g., a cationic ionizable lipid set forth above, DSPC, cholesterol, and ALC-0159.

[0531] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising a cationic ionizable lipid of formula III, DSPC, cholesterol, and ALC-0159.

[0532] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising a cationic ionizable lipid, DSPC, cholesterol, and ALC-0159 as shown in the table above.

[0533] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and ALC-0159.

[0534] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising ALC-0366, DSPC, cholesterol, and ALC-0159.

[0535] In some embodiments, a nucleic acid, such as an RNA, described herein is formulated in an LNP composition comprising ALC-0315, DSPC, cholesterol, and ALC-0159. 3D-P-DMA: (6Z,16Z)-12-((Z)-dec-4-en-1-yl)docosa-6,16-dien-11-yl 5-(dimethylamino)pentanoate [ka] ALC-0366: ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyloctanoate) [ka] ALC-0315: ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) / 6-[N-6-(2-hexyldecanoyloxy)hexyl-N-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate [ka] DMG-PEG2000: [ka]

[0536] PEG 2000-C-DMA: 3-N-[(ω-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxy-propylamine (MPEG-(2kDa)-C-DMA or methoxy-polyethylene glycol-2,3-bis(tetradecyloxy)propylcarbamate(2000)) [ka] (wherein n has an average value in the range of 30 to 60, for example, about 50) ALC-0159: 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide / 2-[2-(ω-methoxy(polyethylene glycol 2000)ethoxy]-N,N-ditetradecylacetamide [ka] DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine [ka] cholesterol: [ka]

[0537] The N / P value is preferably at least about 4. In some embodiments, the N / P value ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N / P value is about 6.

[0538] Cells for targeted delivery According to the present disclosure, the nucleic acid payload is specifically delivered to the target cell by targeting a target on the target cell, e.g., an antigen on the target cell, also referred to herein as the "primary target."

[0539] In some embodiments, the primary target is a structure, such as a protein, present on the surface of the target cell, such as a cell surface antigen, including a cell surface receptor.

[0540] Terms such as "expressed on the cell surface," "associated with the cell surface," or "cell surface molecule" mean that a molecule, such as a receptor or antigen, is located in association with the plasma membrane of a cell, with at least a portion of the molecule facing the extracellular space of the cell and accessible from the outside of the cell by a binding molecule, such as an antibody, located on the outside of the cell. In this context, a portion is preferably at least 4, preferably at least 8, preferably at least 12, more preferably at least 20 amino acids. The association may be direct or indirect. For example, the association may be through one or more transmembrane domains, one or more lipid anchors, or through interaction with any other protein, lipid, sugar, or other structure that can be found on the outer leaflet of the plasma membrane of the cell. For example, a molecule associated with the surface of a cell may be a transmembrane protein having an extracellular portion, or a protein that associates with the surface of a cell by interacting with another protein that is a transmembrane protein.

[0541] "Cell surface" or "surface of a cell" is used according to its ordinary meaning in the art and thus includes the outside of a cell that is accessible to binding by proteins and other molecules. An antigen is expressed on the surface of a cell if it is located on the surface of the cell and is accessible for binding, for example, by an antigen-specific antibody added to the cell. In one embodiment, an antigen expressed on the surface of a cell is an integral membrane protein having an extracellular portion that is recognized by a binding molecule such as an antibody.

[0542] The term "extracellular portion" or "exodomain" in the context of the present invention refers to a part of a molecule, such as a protein, that faces the extracellular space of a cell and that is preferably accessible from outside the cell, e.g. by binding to a molecule, such as an antibody, that is located on the outside of said cell.

[0543] Primary targets may be upregulated during disease, such as infection or cancer. In diseased tissue, the markers differ from healthy tissue, which can offer unique possibilities for treatment, particularly targeted therapy.

[0544] In some embodiments, the primary target is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, allowing diseased cells to be targeted by the methods and agents described herein, for example, to deliver a pharmaceutically active agent.

[0545] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with disease. A disease-associated antigen may be associated with infection by a microorganism, typically a microbial antigen, or may be associated with cancer, typically a tumor.

[0546] In some embodiments, the primary target is a tumor antigen. In the context of the present disclosure, the term "tumor antigen" or "tumor-associated antigen" refers to a protein that is specifically expressed in a limited number of tissues and / or organs or at a specific developmental stage under normal conditions. For example, a tumor antigen may be specifically expressed in gastric tissue, preferably gastric mucosa, reproductive organs, such as the testis, trophoblast tissue, such as the placenta, or germline cells under normal conditions, and is expressed or aberrantly expressed in one or more tumor or cancer tissues. In this context, "a limited number" preferably means 3 or less, more preferably 2 or less. In the context of the present disclosure, tumor antigens include, for example, differentiation antigens, preferably cell type-specific differentiation antigens, i.e., proteins that are specifically expressed in a specific cell type at a specific differentiation stage under normal conditions, cancer / testis antigens, i.e., proteins that are specifically expressed in the testis and sometimes the placenta under normal conditions, and germline-specific antigens. In the context of the present disclosure, tumor antigens are preferably associated with the cell surface of cancer cells and are preferably not expressed or only rarely expressed in normal tissues. Preferably, tumor antigens or aberrant expression of tumor antigens identify cancer cells. In the context of the present disclosure, the tumor antigen expressed by cancer cells in a subject, for example, a patient suffering from cancer disease, is preferably the self-protein of the subject.In a preferred embodiment, in the context of the present disclosure, the tumor antigen is specifically expressed in tissues or organs that are non-essential under normal conditions, that is, tissues or organs that do not cause the death of the subject when damaged by the immune system, or in the organs or structures of the body that are inaccessible or hardly accessible to the immune system.Preferably, the amino acid sequence of the tumor antigen expressed in normal tissues is the same as that of the tumor antigen expressed in cancer tissues.

[0547] Examples of tumor antigens include p53, ART-4, BAGE, beta-catenin / m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, cell surface proteins of the claudin family, such as claudin 6, claudin 18.2 and claudin 12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE E-A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-1 / MelanA, MC1R, myosin / m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 minor BCR-abL, Pm1 / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, survivin, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, and WT. Particularly preferred tumor antigens include claudin 18.2 (CLDN18.2) and claudin 6 (CLDN6).

[0548] In some embodiments, the primary target is a structure, such as a protein, present on the surface of the target cell, such as a cell surface antigen or cell surface receptor, whose presence or amount is characteristic of a particular cell type compared to other cell types. This allows a particular cell type, characterized by its presence or increased amount, to be targeted by the methods and agents described herein. In some embodiments, the cells for targeted delivery are immune effector cells, and the primary target is a cell surface antigen characteristic of immune effector cells. Targeting immune effector cells by the methods and agents described herein allows for the transfection of these cells with a nucleic acid encoding the antigen receptor and the generation of immune effector cells genetically modified to express the antigen receptor.

[0549] immune effector cells Immune effector cells used in connection with the methods and medicaments described herein and into which nucleic acids (DNA or RNA) encoding antigen receptors can be introduced include, in particular, immune effector cells such as cells with lytic capacity, particularly lymphoid cells, preferably T cells, and in particular, cytotoxic lymphocytes, preferably selected from cytotoxic T cells, natural killer (NK) cells, and lymphokine-activated killer (LAK) cells. Upon activation, these cytotoxic lymphocytes each cause target cell destruction. For example, cytotoxic T cells cause target cell destruction by one or both of the following means: First, upon activation, T cells release cytotoxins such as perforin, granzymes, and granulysin. Perforin and granulysin create pores in the target cell, and granzymes enter the cell and trigger a cytoplasmic caspase cascade that induces apoptosis (programmed cell death) of the cell. Second, apoptosis can be induced via Fas-Fas ligand interaction between T cells and target cells. The cells used in connection with the present disclosure are preferably autologous cells, although xenogeneic or allogeneic cells can be used.

[0550] The term "effector function" in the context of the present disclosure includes any function mediated by a component of the immune system that results in the inhibition of tumor growth and / or tumorigenesis, including, for example, the killing of diseased cells, such as tumor cells, or the inhibition of tumor dissemination and metastasis. Preferably, the effector function in the context of the present disclosure is a T cell-mediated effector function. Such a function is mediated by helper T cells (CD4 + T cells), cytokine release and / or CD8 + It involves the activation of lymphocytes (CTLs) and / or B cells, and in the case of CTLs, the elimination of cells, i.e., cells characterized by expression of the antigen, e.g., via apoptosis or perforin-mediated cytolysis, the production of cytokines such as IFN-γ and TNF-α, and the specific cytolytic killing of target cells expressing the antigen.

[0551] The term "immune effector cell" or "immunoreactive cell" in the context of the present disclosure relates to a cell that exerts an effector function during an immune response. In some embodiments, an "immune effector cell" can bind to an antigen, such as an antigen presented in association with MHC on a cell or expressed on the surface of a cell, and mediate an immune response. For example, immune effector cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in the context of the present disclosure, an "immune effector cell" refers to a T cell, preferably a CD4 + and / or CD8 + T cells, most preferably CD8 + The term "immune effector cells" also includes cells that can mature into immune cells (such as T cells, especially T helper cells, or cytolytic T cells) upon appropriate stimulation. Immune effector cells express CD34 + They include hematopoietic stem cells, immature and mature T cells, and immature and mature B cells. The differentiation of T cell precursors into cytolytic T cells resembles the clonal selection of the immune system upon exposure to antigen.

[0552] In some embodiments, the genetically modified immune effector cells are CAR-expressing immune effector cells. In some embodiments, the genetically modified immune effector cells are TCR-expressing immune effector cells.

[0553] Immune effector cells used in the present invention may express an endogenous antigen receptor, such as a T cell receptor or a B cell receptor, or may lack expression of an endogenous antigen receptor.

[0554] "Lymphoid cells" are cells or precursors of such cells that can generate immune responses, such as cellular immune responses, optionally after appropriate modification, for example, after introduction of antigen receptors such as TCRs or CARs, and include lymphocytes, preferably T lymphocytes, lymphoblasts, and plasma cells. Lymphoid cells can be immune effector cells as described herein. Preferred lymphoid cells are T cells that can be modified to express antigen receptors on their cell surface. In some embodiments, lymphoid cells lack endogenous expression of T cell receptors.

[0555] The terms "T cell" and "T lymphocyte" are used interchangeably herein and refer to T helper cells (CD4 + Cytotoxic T cells (CTL, CD8 + T cells).

[0556] T cells belong to a group of white blood cells known as lymphocytes and play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells, by the presence of a special receptor on their cell surface called the T cell receptor (TCR). The thymus is the primary organ responsible for the maturation of T cells. Several different subsets of T cells have been discovered, each with distinct functions.

[0557] T helper cells assist other white blood cells in immunological processes, including, among other functions, the maturation of B cells into plasma cells and the activation of cytotoxic T cells and macrophages. These cells express the CD4 glycoprotein on their surface and are therefore CD4 + Also known as T cells, helper T cells are activated when presented with peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or support the active immune response.

[0558] Cytotoxic T cells destroy virus-infected and tumor cells and are also involved in transplant rejection. These cells express the CD8 glycoprotein on their surface and are therefore known as CD8 + Also known as T cells, these cells recognize their targets by binding to antigens associated with MHC class I, which are present on the surface of almost every cell in the body.

[0559] "Regulatory T cells" or "Tregs" are a subpopulation of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. Tregs are immunosuppressive and generally suppress or downregulate the induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FoxP3, and CD25.

[0560] As used herein, the term "naive T cells" refers to mature T cells that, unlike activated or memory T cells, have never encountered their cognate antigen in the periphery. Naive T cells are generally characterized by surface expression of L-selectin (CD62L), the absence of activation markers CD25, CD44, or CD69, and the absence of the memory CD45RO isoform.

[0561] As used herein, the term "memory T cells" refers to a subgroup or subpopulation of T cells that previously encountered and responded to their cognate antigen. Upon a second encounter with the antigen, memory T cells can regenerate to mount a faster and more potent immune response than the first time the immune system responded to the antigen. Memory T cells are CD4 + or CD8 + and typically express CD45RO.

[0562] As used herein, the term "T cells" also includes cells that can mature into T cells with appropriate stimulation.

[0563] The majority of T cells have a T cell receptor (TCR) that exists as a complex of several proteins. The actual T cell receptor is produced from independent T cell receptor alpha and beta (TCRα and TCRβ) genes and is composed of two separate peptide chains called the α- and β-TCR chains. γδ T cells (gamma delta T cells) are a small subset of T cells that have separate T cell receptors (TCRs) on their surface. However, in γδ T cells, the TCR is composed of one γ chain and one δ chain. This group of T cells is much rarer than αβ T cells (2% of all T cells).

[0564] All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells derived from hematopoietic stem cells reside in the thymus and expand by cell division to generate a large population of immature thymocytes. The earliest thymocytes express neither CD4 nor CD8 and are therefore double-negative (CD4 - CD8 - ) cells. As development progresses, they become double-positive thymocytes (CD4 + CD8 + ) and eventually become single positive (CD4 + CD8 - or CD4 - CD8 + ) mature into thymocytes, which are then released from the thymus into peripheral tissues.

[0565] T cells can generally be prepared in vitro or ex vivo using standard procedures.For example, T cells can be isolated from the bone marrow, peripheral blood, or bone marrow or peripheral blood fraction of mammals such as patients using commercially available cell separation systems.Alternatively, T cells can be derived from related or unrelated humans, non-human animals, cell lines, or cultures.The sample containing T cells can be, for example, peripheral blood mononuclear cells (PBMCs).

[0566] As used herein, the term "NK cells" or "natural killer cells" refers to a subset of peripheral blood lymphocytes defined by expression of CD56 or CD16 and the absence of a T cell receptor. As provided herein, NK cells can also be differentiated from stem or progenitor cells.

[0567] Targeted Delivery of Nucleic Acid Payloads The agents and methods described herein have utility in a variety of applications in which it is desirable to introduce a nucleic acid payload, e.g., an exogenous nucleic acid sequence, into a target cell, and are of particular interest when it is desirable to express a peptide or polypeptide encoded by the nucleic acid in the target cell into which the nucleic acid has been introduced. The agents described herein can be administered by in vitro or in vivo protocols.

[0568] Delivery of nucleic acid payloads, e.g., nucleic acid transfection, using the methods and agents described herein can be used with various target cells so that the nucleic acid payload is introduced into the target cells. The present disclosure can provide for in vitro or in vivo introduction of nucleic acid payloads into target cells, depending on the location of the target cells. For example, if the target cells are isolated cells, the nucleic acid payload can be directly introduced into the cells under cell culture conditions that allow the target cells to survive. Alternatively, if the target cells or cells are part of a multicellular organism, the targeted particles described herein can be administered to the organism or host in a manner that allows the targeted particles to enter the target cells. By "in vivo" is meant that the targeted particles are administered to the living body of an animal. By "ex vivo" is meant that the cells are modified outside the body. Such cells can be reintroduced into living cells. The route of administration of targeted particles to a multicellular organism depends on several parameters, including the nature of the targeted particles. Particularly interesting systemic route is the vascular route, in which targeted particles are introduced into the host's vascular system, for example, artery or vein, and intravenous administration route is particularly interesting in many embodiments.For administration, targeted particles are typically present in pharmaceutical preparations, for example, including pharmaceutically acceptable carriers, diluents and / or adjuvants, and contain an effective amount of payload.In certain embodiments, targeted particles are administered in aqueous delivery vehicles, for example, saline solution.Therefore, in many embodiments, targeted particles are administered into blood vessels, for example, into arteries or into veins, using aqueous delivery vehicles, for example, saline solution.

[0569] In many embodiments, the targeted particle is administered to a multicellular organism in an in vivo manner, such that the nucleic acid payload is introduced into target cells of the multicellular organism. In the case of a nucleic acid payload, administration is typically under conditions sufficient for expression of the nucleic acid to occur. In some embodiments, the agents and methods described herein result in persistent expression of the nucleic acid payload, rather than transient expression, as indicated above. By persistent expression, it is meant that expression of the nucleic acid at detectable levels continues for an extended, if not indefinite, period of time after administration of the nucleic acid payload. By extended period of time, it is meant at least one week, usually at least two months, and more usually at least six months. By detectable level, it is meant that expression of the nucleic acid is at a level such that the encoded protein can be detected in a mammal, for example, in the serum of the mammal, at therapeutic concentrations.

[0570] In some embodiments, the persistent expression described above is achieved with or without the integration of nucleic acid payload into the host's target cell genome.In some embodiments, the nucleic acid introduced into target cell is integrated into the target cell genome, i.e., into one or more chromosomes of target cell.In some embodiments, the nucleic acid is maintained episomally, for example, it is an episomal vector that provides persistent expression.

[0571] Thus, the cells described herein, e.g., immune effector cells, can be genetically modified ex vivo / in vitro or in vivo in the subject being treated to express a peptide or polypeptide, e.g., an antigen receptor, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR)-binding antigen, or its processing product, particularly when present on or presented by target cells, e.g., antigen-presenting cells or diseased cells. In some embodiments, the modification to express a peptide or polypeptide, e.g., an antigen receptor, occurs in vivo. The cells may be endogenous cells of the patient or may have been administered to the patient. In some embodiments, the modification to express a peptide or polypeptide, e.g., an antigen receptor, occurs ex vivo / in vitro. The modified cells can then be administered to the patient.

[0572] In some embodiments, the methods and medicaments described herein are used to transfect immune effector cells with nucleic acids encoding antigen receptors to generate immune effector cells genetically modified to express the antigen receptor.

[0573] T cell receptor (TCR) As used herein, the term "T cell receptor" or "TCR" refers to a protein receptor on T cells composed of a heterodimer of alpha (α) and beta (β) chains, although in some cells the TCR consists of gamma and delta (γδ) chains. In some embodiments, the TCR can be derived from any cell that contains a TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and gamma delta T cells. Each α, β, γ, and δ chain is composed of two Ig-like domains: a variable domain (V) that confers antigen recognition via complementarity-determining regions (CDRs), followed by a constant domain (C) that is anchored to the cell membrane by a connecting peptide and a transmembrane (TM) region. The TM region associates with the invariant subunit of the CD3 signaling apparatus. Each V domain has three CDRs. These CDRs interact with the complex between antigen peptides bound to proteins encoded by the major histocompatibility complex (MHC).

[0574] Chimeric antigen receptor (CAR) Adoptive cell transfer therapy using CAR-engineered T cells expressing chimeric antigen receptors is a promising anticancer treatment because CAR-modified T cells can be engineered to target virtually any tumor antigen, preferably in an MHC-independent manner. For example, a patient's T cells can be genetically engineered to express a CAR that is specifically directed against an antigen on the patient's tumor cells.

[0575] As used herein, the term "CAR" (or "chimeric antigen receptor") is synonymous with the terms "chimeric T cell receptor" and "artificial T cell receptor" and relates to an artificial receptor comprising a single molecule or complex of molecules that can recognize, i.e., bind to, a target structure (e.g., an antigen) on a target cell, such as a cancer cell (e.g., by binding of an antigen-binding domain to an antigen expressed on the surface of the target cell), and confer specificity to an immune effector cell, such as a T cell, that expresses the CAR on its cell surface. Such cells do not necessarily require antigen processing and presentation for target cell recognition, but rather can preferably specifically recognize any antigen present on the target cell. Preferably, recognition of the target structure by a CAR results in activation of the immune effector cell that expresses the CAR. A CAR can comprise one or more protein units comprising one or more domains described herein. The term "CAR" does not include T cell receptors.

[0576] CARs generally comprise a target-specific binding element, also referred to as an antigen-binding portion or domain, which is part of the extracellular domain of the CAR. In particular, CARs can target antigens on target cells, e.g., diseased cells such as tumor cells.

[0577] In some embodiments, the antigen-binding domain comprises a variable region (VH) of an immunoglobulin heavy chain having specificity for an antigen and a variable region (VL) of an immunoglobulin light chain having specificity for an antigen. In some embodiments, the immunoglobulin is an antibody. In some embodiments, the heavy chain variable region (VH) and the corresponding light chain variable region (VL) are connected via a peptide linker. Preferably, part of the antigen-binding portion in the CAR is an scFv. In some embodiments, the antigen-binding domain comprises a VHH domain.

[0578] CAR is preferably designed to include a transmembrane domain fused to the extracellular domain of CAR.In some embodiments, the transmembrane domain is not naturally associated with one of the domains in CAR.In some embodiments, the transmembrane domain is naturally associated with one of the domains in CAR.In some embodiments, the transmembrane domain is modified by amino acid substitution to prevent such domain from binding to the transmembrane domain of the same or different surface membrane protein, thereby minimizing interaction with other members of the receptor complex.The transmembrane domain can be derived from either natural or synthetic sources.If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane region particularly used herein may be derived from (i.e., comprise at least one or more transmembrane regions of) the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domain may be synthetic, in which case it comprises primarily hydrophobic residues such as leucine and valine. Preferably, a phenylalanine, tryptophan, and valine triplet is found at each end of the synthetic transmembrane domain.

[0579] In some cases, the CAR comprises a hinge domain that forms a link between the transmembrane domain and the extracellular domain.

[0580] The cytoplasmic domain or other intracellular signaling domain of a CAR is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is placed. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. While the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, as long as it transmits the effector function signal. Thus, the term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0581] It is known that signals generated solely through the TCR are insufficient for full activation of T cells, and that secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and those that act antigen-independently to provide secondary or costimulato...

Claims

1. formula: L-X1-P-X2-B A compound of the formula: During the ceremony, P comprises a polymer; L comprises a hydrophobic moiety attached to said polymer; B comprises a moiety attached to said polymer selected from the group consisting of an antibody or antibody-like molecule that binds to a cell surface antigen, an ALFA tag, and a moiety that binds to an ALFA tag; X1 is absent or a first linking moiety; and X2 is absent or a second linking moiety; compound.

2. (A) the hydrophobic moiety comprises a moiety selected from vitamin E, dialkylamines, diacylglycerides and ceramides, two C8-C24 hydrocarbon chains, lipids, and / or phospholipids; and / or a moiety selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine), and mixtures thereof; (B) the polymer is a hydrophilic polymer or is 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), and combinations thereof; and / or (C) X2 comprises the reaction product of a thiol or cysteine ​​reactive group with a thiol or cysteine ​​group of the compound comprising moiety B; The compound of claim 1.

3. The compound of claim 2 , wherein the thiol or cysteine ​​reactive group comprises a maleimide group. Claim 4: The compound comprises the reaction product of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)] and a compound having the formula SH(CH2)nC(O)-B, where n is in the range of 1 to 5; or The compound is formula 【Chemistry 1】 4. The compound of claim 3, wherein 5. The compound of claim 4, wherein n is 2.

6. The compound of claim 1 , wherein the antibody-like molecule comprises an antibody fragment or a DARPin.

7. 2. The compound of claim 1, wherein the cell surface antigen is selected from the group consisting of CD4, CD8, and CD3.

8. A functionalized particle comprising one or more particle-forming components, a nucleic acid payload, and the compound of claim 1, wherein the compound is incorporated into the particle via the hydrophobic moiety.

9. B comprises a moiety that binds to an AlFA tag or an ALFA tag, and the functionalized particle has the formula: B'-X3-B'' (In the formula, B' comprises a moiety that binds to B; X3 is absent or a linking moiety; and B'' comprises a portion that binds to a cell surface antigen) further comprising the compound of The functionalized particle of claim 8 .

10. 10. The functionalized particle of claim 9, wherein the compound of the formula B'-X3-B'' comprises a peptide or polypeptide.

11. B comprises an ALFA tag and B' comprises a moiety that binds to said ALFA tag; or B' comprises an ALFA tag and B comprises a moiety that binds to said ALFA tag; The functionalized particle of claim 8 .

12. The functionalized particle of claim 11, wherein the portion that binds to the ALFA tag comprises an antibody or antibody-like molecule.

13. The functionalized particle of claim 12 , wherein the antibody-like molecule comprises an antibody fragment or a DARPin.

14. The functionalized particle of claim 11 , wherein the portion that binds to the ALFA tag comprises a VHH domain comprising the CDR1 sequence VTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY.

15. (i) The particle is a lipid particle, a polymer particle, or a mixture thereof. (ii) the particle is a non-viral particle, or (iii) the particles are nanoparticles and / or the nucleic acids comprise DNA and / or RNA; The functionalized particle of claim 8 .

16. The functionalized particle of claim 8 , wherein the nucleic acid comprises a nucleic acid encoding an antigen receptor.

17. 17. The functionalized particle of claim 16, wherein the antigen receptor comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

18. An ex vivo method for delivering nucleic acids to cells expressing a cell surface antigen, the method comprising adding to the cells a composition comprising the functionalized particles of claim 8.

19. 20. The method of claim 18, wherein the cells comprise immune effector cells.

20. the immune effector cells comprise T cells, or 20. The method of claim 19, wherein the immune effector cells comprise CD8+ and / or CD4+ T cells.

21. a method for preparing genetically modified cells, or 19. The method of claim 18, which is a method for preparing immune effector cells genetically modified to express an antigen receptor.

22. the genetic modification is transient or stable; or 22. The method of claim 21, wherein the genetic modification is carried out by a viral-based method, a transposon-based method, or a gene editing-based method.

23. A pharmaceutical composition comprising the functionalized particle of any one of claims 8 to 17 for use in a method for preparing immune effector cells genetically modified to express an antigen receptor, comprising: The method comprises administering the composition to a subject having immune effector cells, wherein the nucleic acid comprises a nucleic acid encoding an antigen receptor.

24. 24. The pharmaceutical composition of claim 23, wherein B or B'' comprises a moiety that binds to a cell surface antigen on said immune effector cell.

25. the immune effector cells comprise T cells, or the immune effector cells comprise CD8+ and / or CD4+ T cells, and / or 24. The pharmaceutical composition of claim 23, wherein the cell surface antigen is selected from the group consisting of CD4, CD8 and CD3.

26. 24. The pharmaceutical composition of claim 23, wherein the antigen receptor comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

27. A pharmaceutical composition comprising immune effector cells genetically modified to express an antigen receptor for use in a method of treating a subject, comprising: The method comprises: (I) preparing the immune effector cells ex vivo using the method according to any one of claims 18 to 22; and (II) administering the immune effector cells to the subject. A pharmaceutical composition comprising:

28. A pharmaceutical composition comprising the functionalized particle of any one of claims 8 to 17 for use in a method of treating a subject, comprising: The method includes administering the composition to a subject having immune effector cells, wherein the nucleic acid includes a nucleic acid encoding an antigen receptor. Pharmaceutical compositions.

Images