Agents and methods for targeted delivery of cytokines to immune cells

Abstract

The invention relates to agents and methods for targeted delivery of RNA such as mRNA encoding a polypeptide comprising a cytokine ora functional variant thereof to immune cells for expression of the polypeptide. Delivering RNA encoding a cytokine to immune cells may be useful for immunomodulation of immune cells, in particular for inducing proliferation of immune cells. In some embodiments, the invention involves a particle, and a targeting compound comprising a moiety incorporating into the particle, e.g., a hydrophobic moiety, and having a binding moiety covalently attached thereto. The particle carries an RNA payload, i.e., RNA encoding a polypeptide comprising a cytokine or a functional variant thereof. Targeting of an immune cell may be achieved by the direct or indirect binding of the targeting compound to cell surface antigens on the target immune cell of interest.

Description

[0001] AGENTS AND METHODS FOR TARGETED DELIVERY OF CYTOKINES TO IMMUNE CELLS

[0002] The invention relates to agents and methods for targeted delivery of RNA such as mRNA encoding a polypeptide comprising a cytokine or a functional variant thereof to immune cells for expression of the polypeptide. Delivering RNA encoding a cytokine to immune cells may be useful for immunomodulation of immune cells, in particular for inducing proliferation of immune cells. In some embodiments, the invention involves a particle, and a targeting compound comprising a moiety incorporating into the particle, e.g., a hydrophobic moiety, and having a binding moiety covalently attached thereto. The particle carries an RNA payload, i.e., RNA encoding a polypeptide comprising a cytokine or a functional variant thereof. Targeting of an immune cell may be achieved by the direct or indirect binding of the targeting compound to cell surface antigens on the target immune cell of interest.

[0003] In some embodiments, the binding moiety of the targeting compound binds to a cell surface antigen on a target immune cell, e.g., an immune effector cell, for targeting the particle carrying an RNA payload to target immune cells. In these embodiments, the binding moiety of the targeting compound may be constructs that have affinity for cell surface targets, e.g., membrane proteins, and include antibodies or antibody fragments.

[0004] In some embodiments, the binding moiety of the targeting compound binds to a docking compound binding to a cell surface antigen on a target immune cell, e.g., an immune effector cell, for targeting the particle carrying an RNA payload to target immune cells. In some embodiments, the docking compound comprises a peptide or polypeptide. In some embodiments, the docking compound comprises a binding moiety binding to target immune cells (primary targeting moiety) and a further binding moiety binding to the binding moiety of the targeting compound. The binding moiety of the targeting compound may bind to its binding moiety on the docking compound and then the primary targeting moiety may bind to a target antigen on target immune cells such as an antigen on immune effector cells to thereby precisely deliver an RNA payload to the target immune cells such as immune effector cells.

[0005] The present invention relates to an approach wherein particles comprising an RNA payload and a targeting compound are used. In some embodiments, the targeting compound comprises (i) a hydrophobic moiety for incorporation into the particles and (ii) a binding moiety covalently attached to the hydrophobic moiety for direct or indirect targeting of the particles to target immune cells and delivering the RNA payload to target immune cells. Cell targeting may be achieved by the direct or indirect binding of the targeting compound to cell surface antigens on the target immune cell of interest.

[0006] In some embodiments, particles comprising an RNA payload and a targeting compound are administered. In some embodiments, the binding moiety of the targeting compound binds to target immune cells, e.g., by binding to a cell surface antigen, thus resulting in cellular uptake of the RNA payload. Common examples for target immune cell binding moieties on the targeting compound are antibodies.

[0007] In some embodiments, particles comprising an RNA payload and a targeting compound, and a docking compound that binds to target immune cells, e.g., by binding to a cell surface antigen, are administered. In these embodiments, the targeting compound may be equipped with a binding moiety targeting a moiety on the docking compound. In some embodiments, a docking compound which is bound via a targeting compound to a particle comprising an RNA payload is administered. The docking compound may bind to target immune cells, e.g., by bindingto a cell surface antigen, thus resulting in cellular uptake of the RNA payload. Common examples for pairs of interacting moieties on the targeting compound and on the docking compound are antibody / antigen systems. Common examples for target immune cell binding moieties on the docking compound are antibodies.

[0008] Summary

[0009] The invention relates to agents and methods for targeted delivery of RNA encoding a polypeptide comprising a cytokine or a functional variant thereof, i.e., an RNA payload, to immune cells. The agents and methods for targeted delivery of an RNA payload described herein may be used for generating in vitro / ex vivo or in vivo immune cells, e.g., immune effector cells, transfected with RNA encoding a polypeptide comprising a cytokine or a functional variant thereof. In some embodiments, immune cells transfected with RNA encoding a polypeptide comprising a cytokine or a functional variant thereof express the polypeptide. Transfection is achieved using particles described herein comprising RNA encoding a polypeptide comprising a cytokine or a functional variant thereof and a targeting compound for targeting immune cells directly or via a docking compound binding to the targeting compound. The particles may deliver the RNA to immune cells in vitro / ex vivo as well as in vivo. Immune effector cells transfected as described herein are useful in the treatment of diseases wherein targeting cells such as diseased cells expressing an antigen such as a tumor antigen is beneficial. The target cells may express the antigen on the cell surface for recognition by a CAR or in the context of MHC for recognition by a TCR. The treatments described herein may provide for the selective eradication of such cells expressing an antigen, thereby minimizing adverse effects to normal cells not expressing the antigen. Immune effector cells expressing an antigen receptor, e.g., a CAR or TCR, targeting cells through binding to the antigen (or a procession product thereof) may be provided to a subject such as by administration of genetically modified immune effector cells to the subject or generation of genetically modified immune effector cells in the subject. 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 on the 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 are, in particular, useful for the treatment of diseases characterized by diseased cells expressing an antigen the immune effector cells are directed to. In some embodiments, the immune effector cells by means of a CAR have a binding specificity for disease-associated antigen when present on diseased cells. In some embodiments, the immune effector cells by means of a TCR have a binding specificity for a procession product of disease-associated antigen when presented on diseased cells. In some embodiments, an immune effector cell is genetically modified to stably or transiently express an antigen receptor on its surface.

[0010] In one aspect, the invention relates to a particle comprising:

[0011] (a) one or more particle forming components, and

[0012] (b) a targeting compound comprising:

[0013] (i) a moiety incorporating the targeting compound into the particle, and

[0014] (ii) a moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag, wherein the particle carries RNA encoding a polypeptide comprising a cytokine or a functional variant thereof.

[0015] In some embodiments, the moiety incorporating the targeting compound into the particle, and the moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag are linked by a linking moiety comprising a polymer P.

[0016] In some embodiments, the targeting compound comprises the formula:

[0017] L-X1-P-X2-B wherein

[0018] P is absent or comprises a polymer,

[0019] L comprises a moiety incorporating the targeting compound into the particle,

[0020] B comprises a moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag,

[0021] XI is absent or a first linking moiety, and

[0022] X2 is absent or a second linking moiety.

[0023] In some embodiments, the particle comprises a lipid particle, a polymer particle, or a mixture thereof. In some embodiments, the particle comprises a lipid particle.

[0024] In some embodiments, the moiety incorporating the targeting compound into the particle comprises a hydrophobic moiety

[0025] In some embodiments, the moiety incorporating the targeting compound into the particle comprises a moiety selected from vitamin E, dialkylamine, diacylglyceride and ceramide.

[0026] In some embodiments, the moiety incorporating the targeting compound into the particle comprises a C8-C24 hydrocarbon chain. In some embodiments, the moiety incorporating the targeting compound into the particle comprises two C8-C24 hydrocarbon chains.

[0027] In some embodiments, the moiety incorporating the targeting compound into the particle comprises a lipid.

[0028] In some embodiments, the moiety incorporating the targeting compound into the particle comprises a phospholipid.

[0029] In some embodiments, the moiety incorporating the targeting compound into the particle comprises a moiety selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine), and mixtures thereof.

[0030] In some embodiments, the targeting compound is incorporated into the particle through a charge in the moiety incorporating the targeting compound into the particle interacting with an opposite charge in the particle. In some embodiments, the particle has a positive charge and the targeting compound is incorporated into the particle through a negative charge in the moiety incorporating the targeting compound into the particle interacting with the positive charge of the particle. In some embodiments, the one or more particle forming components comprise a polymer. In some embodiments, the one or more particle forming components comprise a polymer having a net positive charge. In some embodiments, the one or more particle forming components comprise a polymer comprising one or more ionizable nitrogen atoms. In some embodiments, the moiety incorporating the targeting compound into the particle comprises a polymer. In some embodiments, the moiety incorporating the targeting compound into the particle comprises a polymer having a net negative charge. In some embodiments, the moiety incorporating the targeting compound into the particle comprises a polymer comprising one or more ionizable carboxy groups. In some embodiments, the moiety incorporating the targeting compound into the particle comprises a polyglutamic acid moiety.

[0031] In some embodiments, P comprises a hydrophilic polymer.

[0032] In some embodiments, P is selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), derivatives and combinations thereof.

[0033] 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 the moiety B.

[0034] In some embodiments, the thiol or cysteine reactive group comprises a maleimide group.

[0035] In some embodiments, the targeting compound comprises the reaction product of 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)] with a compound comprising the formula SH(CH2)nC(O)-B, wherein n ranges from 1 to 5 and preferably n is 2.

[0036] In some embodiments, the targeting compound comprises a compound of the formula:

[0037] In some embodiments, the moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag comprises a moiety binding to a cell surface antigen on immune cells.

[0038] In some embodiments, the moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag comprises a moiety selected from the group consisting of a tag and a moiety binding to a tag and the particle further comprises a docking compound comprising:

[0039] (i) a moiety binding to the moiety selected from the group consisting of a tag and a moiety binding to a tag, and

[0040] (ii) a moiety binding to a cell surface antigen on immune cells.

[0041] In some embodiments, the docking compound comprises the formula:

[0042] B'-X3-B" wherein

[0043] B' comprises a moiety binding to the moiety selected from the group consisting of a tag and a moiety binding to a tag,

[0044] X3 is absent or a linking moiety, and

[0045] B" comprises a moiety binding to a cell surface antigen on immune cells.

[0046] In some embodiments, the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a tag and the moiety binding to the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a moiety binding to the tag.

[0047] In some embodiments, the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a moiety binding to a tag and the moiety binding to the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a tag to which the moiety binding to a tag binds.

[0048] In some embodiments, the docking compound comprises a peptide or polypeptide.

[0049] In some embodiments, the moiety bindingto a cell surface antigen on immune cells comprises a peptide or polypeptide.

[0050] In some embodiments, the moiety bindingto a cell surface antigen on immune cells comprises an antibody or antibody-like molecule.

[0051] In some embodiments, the antibody-like molecule comprises an antibody fragment or DARPin.

[0052] In some embodiments, the antibody-like molecule comprises a nanobody.

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

[0054] In some embodiments, the immune cells comprise CD8+ and / or CD4+ T cells.

[0055] In some embodiments, the cell surface antigen on immune cells is characteristic for the immune cells.

[0056] In some embodiments, the cell surface antigen on immune cells is selected from the group consisting of CD4, CD8, CD3, CD2, CD5, and CD127. In some embodiments, the cell surface antigen on immune cells is selected from the group consisting of CD4, CD8 and CD3 In some embodiments, the moiety binding to a tag comprises a peptide or polypeptide.

[0057] In some embodiments, the moiety binding to a tag comprises an antibody or antibody-like molecule. In some embodiments, the antibody-like molecule comprises an antibody fragment or DARPin.

[0058] In some embodiments, the antibody-like molecule comprises a nanobody.

[0059] In some embodiments, the tag comprises a peptide or polypeptide.

[0060] In some embodiments, the tag comprises a peptide tag.

[0061] In some embodiments, the tag comprises an ALFA-tag.

[0062] In some embodiments, the tag comprises an ALFA-tag and the moiety binding to the tag comprises a VHH domain comprising the CDR1 sequence VTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY.

[0063] In some embodiments, the particle is a non-viral particle.

[0064] In some embodiments, the particle is a nanoparticle.

[0065] In some embodiments, the particle is a lipid nanoparticle (LNP).

[0066] In some embodiments, the cytokine comprises an interleukin.

[0067] In some embodiments, the cytokine comprises interleukin 2.

[0068] In a further aspect, the invention relates to a composition, e.g., a pharmaceutical composition, comprising particles as described herein.

[0069] In a further aspect, the invention relates to a method for delivering a polypeptide comprising a cytokine or a functional variant thereof to immune cells expressing a cell surface antigen, comprising adding to the immune cells a composition comprising particles, wherein a particle comprises:

[0070] (a) one or more particle forming components, and

[0071] (b) a targeting compound comprising:

[0072] (i) a moiety incorporating the targeting compound into the particle, and

[0073] (ii) a moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag, wherein the particle carries RNA encoding a polypeptide comprising a cytokine or a functional variant thereof. In some embodiments, the moiety incorporating the targeting compound into the particle, and the moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag are linked by a linking moiety comprising a polymer P.

[0074] In some embodiments, the targeting compound comprises the formula:

[0075] L-X1-P-X2-B wherein

[0076] P is absent or comprises a polymer,

[0077] L comprises a moiety incorporating the targeting compound into the particle,

[0078] B comprises a moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag,

[0079] XI is absent or a first linking moiety, and

[0080] X2 is absent or a second linking moiety.

[0081] In some embodiments, the particle comprises a lipid particle, a polymer particle, or a mixture thereof.

[0082] In some embodiments, the particle comprises a lipid particle.

[0083] In some embodiments, the moiety incorporating the targeting compound into the particle comprises a hydrophobic moiety

[0084] In some embodiments, the moiety incorporating the targeting compound into the particle comprises a moiety selected from vitamin E, dialkylamine, diacylglyceride and ceramide.

[0085] In some embodiments, the moiety incorporating the targeting compound into the particle comprises a C8-C24 hydrocarbon chain. In some embodiments, the moiety incorporating the targeting compound into the particle comprises two C8-C24 hydrocarbon chains.

[0086] In some embodiments, the moiety incorporating the targeting compound into the particle comprises a lipid.

[0087] In some embodiments, the moiety incorporating the targeting compound into the particle comprises a phospholipid.

[0088] In some embodiments, the moiety incorporating the targeting compound into the particle comprises a moiety selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine), and mixtures thereof.

[0089] In some embodiments, the targeting compound is incorporated into the particle through a charge in the moiety incorporating the targeting compound into the particle interacting with an opposite charge in the particle. In some embodiments, the particle has a positive charge and the targeting compound is incorporated into the particle through a negative charge in the moiety incorporating the targeting compound into the particle interacting with the positive charge of the particle. In some embodiments, the one or more particle forming components comprise a polymer. In some embodiments, the one or more particle forming components comprise a polymer having a net positive charge. In some embodiments, the one or more particle forming components comprise a polymer comprising one or more ionizable nitrogen atoms. In some embodiments, the moiety incorporating the targeting compound into the particle comprises a polymer. In some embodiments, the moiety incorporating the targeting compound into the particle comprises a polymer having a net negative charge. In some embodiments, the moiety incorporating the targeting compound into the particle comprises a polymer comprising one or more ionizable carboxy groups. In some embodiments, the moiety incorporating the targeting compound into the particle comprises a polyglutamic acid moiety.

[0090] In some embodiments, P comprises a hydrophilic polymer.

[0091] In some embodiments, P is selected from the group consisting of poly(ethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), derivatives and combinations thereof. 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 the moiety B.

[0092] In some embodiments, the thiol or cysteine reactive group comprises a maleimide group.

[0093] In some embodiments, the targeting compound comprises the reaction product of 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)] with a compound comprising the formula SH(CH2)nC(O)-B, wherein n ranges from 1 to 5 and preferably n is 2.

[0094] In some embodiments, the targeting compound comprises a compound of the formula:

[0095] In some embodiments, the moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag comprises a moiety binding to a cell surface antigen on immune cells.

[0096] In some embodiments, the moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag comprises a moiety selected from the group consisting of a tag and a moiety binding to a tag and the particle further comprises a docking compound comprising:

[0097] (i) a moiety binding to the moiety selected from the group consisting of a tag and a moiety binding to a tag, and

[0098] (ii) a moiety binding to a cell surface antigen on immune cells.

[0099] In some embodiments, the docking compound comprises the formula:

[0100] B'-X3-B" wherein

[0101] B' comprises a moiety binding to the moiety selected from the group consisting of a tag and a moiety binding to a tag,

[0102] X3 is absent or a linking moiety, and

[0103] B" comprises a moiety binding to a cell surface antigen on immune cells.

[0104] In some embodiments, the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a tag and the moiety binding to the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a moiety binding to the tag.

[0105] In some embodiments, the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a moiety binding to a tag and the moiety binding to the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a tag to which the moiety binding to a tag binds.

[0106] In some embodiments, the docking compound comprises a peptide or polypeptide.

[0107] In some embodiments, the moiety binding to a cell surface antigen on immune cells comprises a peptide or polypeptide. In some embodiments, the moiety bindingto a cell surface antigen on immune cells comprises an antibody or antibody-like molecule.

[0108] In some embodiments, the antibody-like molecule comprises an antibody fragment or DARPin.

[0109] In some embodiments, the antibody-like molecule comprises a nanobody

[0110] In some embodiments, the moiety binding to a cell surface antigen on immune cells binds to the cell surface antigen expressed by the immune cells.

[0111] In some embodiments, the immune cells comprise T cells.

[0112] In some embodiments, the immune cells comprise CD8+ and / or CD4+ T cells.

[0113] In some embodiments, the cell surface antigen on immune cells is characteristic for the immune cells.

[0114] In some embodiments, the cell surface antigen on immune cells is selected from the group consisting of CD4, CD8, CD3, CD2, CD5, and CD127. In some embodiments, the cell surface antigen on immune cells is selected from the group consisting of CD4, CD8 and CD3 In some embodiments, the moiety binding to a tag comprises a peptide or polypeptide.

[0115] In some embodiments, the moiety binding to a tag comprises an antibody or antibody-like molecule.

[0116] In some embodiments, the antibody-like molecule comprises an antibody fragment or DARPin.

[0117] In some embodiments, the antibody-like molecule comprises a nanobody.

[0118] In some embodiments, the tag comprises a peptide or polypeptide.

[0119] In some embodiments, the tag comprises a peptide tag.

[0120] In some embodiments, the tag comprises an ALFA-tag.

[0121] In some embodiments, the tag comprises an ALFA-tag and the moiety binding to the tag comprises a VHH domain comprising the CDR1 sequence VTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY.

[0122] In some embodiments, the particle is a non-viral particle.

[0123] In some embodiments, the particle is a nanoparticle.

[0124] In some embodiments, the particle is a lipid nanoparticle (LNP).

[0125] In some embodiments, the cytokine comprises an interleukin. In some embodiments, the cytokine comprises interleukin 2.

[0126] In some embodiments, the method is a method for immunomodulation of immune cells.

[0127] In some embodiments, the method is a method for inducing proliferation of immune cells.

[0128] In some embodiments, the immune cells are present ex vivo or in vitro.

[0129] In some embodiments, the immune cells are present in a subject and the method comprises administering the composition to the subject.

[0130] In a further aspect, the invention relates to a method for inducing proliferation of immune cells, comprising adding to the immune cells a composition comprising particles, wherein a particle comprises:

[0131] (a) one or more particle forming components, and

[0132] (b) a targeting compound comprising:

[0133] (i) a moiety incorporating the targeting compound into the particle, and

[0134] (ii) a moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag, wherein the particle carries RNA encoding a polypeptide comprising a cytokine or a functional variant thereof.

[0135] In some embodiments, the moiety incorporating the targeting compound into the particle, and the moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag are linked by a linking moiety comprising a polymer P.

[0136] In some embodiments, the targeting compound comprises the formula:

[0137] L-X1-P-X2-B wherein

[0138] P is absent or comprises a polymer,

[0139] L comprises a moiety incorporating the targeting compound into the particle,

[0140] B comprises a moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag,

[0141] XI is absent or a first linking moiety, and

[0142] X2 is absent or a second linking moiety. In some embodiments, the particle comprises a lipid particle, a polymer particle, or a mixture thereof.

[0143] In some embodiments, the particle comprises a lipid particle.

[0144] In some embodiments, the moiety incorporating the targeting compound into the particle comprises a hydrophobic moiety

[0145] In some embodiments, the moiety incorporating the targeting compound into the particle comprises a moiety selected from vitamin E, dialkylamine, diacylglyceride and ceramide.

[0146] In some embodiments, the moiety incorporating the targeting compound into the particle comprises a C8-C24 hydrocarbon chain. In some embodiments, the moiety incorporating the targeting compound into the particle comprises two C8-C24 hydrocarbon chains.

[0147] In some embodiments, the moiety incorporating the targeting compound into the particle comprises a lipid.

[0148] In some embodiments, the moiety incorporating the targeting compound into the particle comprises a phospholipid.

[0149] In some embodiments, the moiety incorporating the targeting compound into the particle comprises a moiety selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine), and mixtures thereof.

[0150] In some embodiments, the targeting compound is incorporated into the particle through a charge in the moiety incorporating the targeting compound into the particle interacting with an opposite charge in the particle. In some embodiments, the particle has a positive charge and the targeting compound is incorporated into the particle through a negative charge in the moiety incorporating the targeting compound into the particle interacting with the positive charge of the particle. In some embodiments, the one or more particle forming components comprise a polymer. In some embodiments, the one or more particle forming components comprise a polymer having a net positive charge. In some embodiments, the one or more particle forming components comprise a polymer comprising one or more ionizable nitrogen atoms. In some embodiments, the moiety incorporating the targeting compound into the particle comprises a polymer. In some embodiments, the moiety incorporating the targeting compound into the particle comprises a polymer having a net negative charge. In some embodiments, the moiety incorporating the targeting compound into the particle comprises a polymer comprising one or more ionizable carboxy groups. In some embodiments, the moiety incorporating the targeting compound into the particle comprises a polyglutamic acid moiety.

[0151] In some embodiments, P comprises a hydrophilic polymer.

[0152] In some embodiments, P is selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), derivatives and combinations thereof. 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 the moiety B.

[0153] In some embodiments, the thiol or cysteine reactive group comprises a maleimide group.

[0154] In some embodiments, the targeting compound comprises the reaction product of 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)] with a compound comprising the formula SH(CH2)nC(O)-B, wherein n ranges from 1 to 5 and preferably n is 2.

[0155] In some embodiments, the targeting compound comprises a compound of the formula:

[0156] In some embodiments, the moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag comprises a moiety binding to a cell surface antigen on immune cells.

[0157] In some embodiments, the moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag comprises a moiety selected from the group consisting of a tag and a moiety binding to a tag and the particle further comprises a docking compound comprising:

[0158] (i) a moiety binding to the moiety selected from the group consisting of a tag and a moiety binding to a tag, and

[0159] (ii) a moiety binding to a cell surface antigen on immune cells. In some embodiments, the docking compound comprises the formula:

[0160] B'-X3-B" wherein

[0161] B' comprises a moiety binding to the moiety selected from the group consisting of a tag and a moiety binding to a tag,

[0162] X3 is absent or a linking moiety, and

[0163] B" comprises a moiety binding to a cell surface antigen on immune cells.

[0164] In some embodiments, the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a tag and the moiety binding to the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a moiety binding to the tag.

[0165] In some embodiments, the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a moiety binding to a tag and the moiety binding to the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a tag to which the moiety binding to a tag binds.

[0166] In some embodiments, the docking compound comprises a peptide or polypeptide.

[0167] In some embodiments, the moiety binding to a cell surface antigen on immune cells comprises a peptide or polypeptide.

[0168] In some embodiments, the moiety binding to a cell surface antigen on immune cells comprises an antibody or antibody-like molecule.

[0169] In some embodiments, the antibody-like molecule comprises an antibody fragment or DARPin.

[0170] In some embodiments, the antibody-like molecule comprises a nanobody.

[0171] In some embodiments, the moiety binding to a cell surface antigen on immune cells binds to a cell surface antigen expressed by the immune cells.

[0172] In some embodiments, the immune cells comprise T cells.

[0173] In some embodiments, the immune cells comprise CD8+ and / or CD4+ T cells.

[0174] In some embodiments, the cell surface antigen on immune cells is characteristic for the immune cells. In some embodiments, the cell surface antigen on immune cells is selected from the group consisting of CD4, CD8, CD3, CD2, CD5, and CD127. In some embodiments, the cell surface antigen on immune cells is selected from the group consisting of CD4, CD8 and CDS In some embodiments, the moiety binding to a tag comprises a peptide or polypeptide.

[0175] In some embodiments, the moiety binding to a tag comprises an antibody or antibody-like molecule.

[0176] In some embodiments, the antibody-like molecule comprises an antibody fragment or DARPin.

[0177] In some embodiments, the antibody-like molecule comprises a nanobody.

[0178] In some embodiments, the tag comprises a peptide or polypeptide.

[0179] In some embodiments, the tag comprises a peptide tag.

[0180] In some embodiments, the tag comprises an ALFA-tag.

[0181] In some embodiments, the tag comprises an ALFA-tag and the moiety binding to the tag comprises a VHH domain comprising the CDR1 sequence VTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY.

[0182] In some embodiments, the particle is a non-viral particle.

[0183] In some embodiments, the particle is a nanoparticle.

[0184] In some embodiments, the particle is a lipid nanoparticle (LNP).

[0185] In some embodiments, the cytokine comprises an interleukin.

[0186] In some embodiments, the cytokine comprises interleukin 2.

[0187] In some embodiments, the immune cells are present ex vivo or in vitro.

[0188] In some embodiments, the immune cells are present in a subject and the method comprises administering the composition to the subject.

[0189] In a further aspect, the invention relates to a method for treating a subject comprising:

[0190] (i) preparing ex vivo immune cells using the method described herein, and

[0191] (ii) administering the immune cells to the subject.

[0192] In a further aspect, the invention relates to a method for treating a subject comprising administering to the subject a composition comprising particles described herein. In a further aspect, the invention relates to an agent or composition described herein for use in a method for treating a subject described herein.

[0193] Brief description of the drawings

[0194] Figure 1: Physicochemical characterization of DODMA-based lipid nanoparticles (LNPs) containing Thyl.l / hlL-2 mRNA mix. LNPs were formulated with an RNA mix of 1:1 w / w, with or without ahCD3 VHH X NbALFA ligand. Size (A) and PDI (B) were in the expected range, measured via dynamic light scattering. Successful RNA incorporation was verified via agarose gel electrophoresis (not shown). n=3 technical replicates / group.

[0195] Figure 2: In vitro transfection efficiency of lipid nanoparticles (LNPs) containing Thyl.l / hlL-2 mRNA mix. Human PBMCs were transfected with respective LNPs (2000 ng RNA total; 250 ng Thyl.l RNA (A), 1750 ng hlL-2 RNA (B); RNA mix 1:8 VJ / VJ; LNPS were formulated with or without ahCD3 VHH X NbALFA ligand). PMA / lono was supplemented to medium as positive control, lx HBT buffer (10 mM HEPES, 10 w / v % Trehalose, pH 7.1) served as negative control. %Thyl.l- and %h IL- 2 expressing lymphocyte sub-populations analyzed by flow cytometry. n=3 technical replicates / group.

[0196] Figure 3: Physicochemical characterization of HY501-based lipid nanoparticles (LNPs) containing Thyl.l / Luc and Thyl.l / hlL-2 mRNA mix. Respective LNPs were formulated with an RNA mix of 1:1 w / w with or without ahCD3 VHH X NbALFA ligand. Size (A) and PDI (B) measured via dynamic light scattering. RNA integrity performed by Fragment Analyzer (C). Zeta Potential measured via Zetasizer (D). All physicochemical parameters in the expected range. Successful RNA incorporation was verified via agarose gel electrophoresis (not shown).

[0197] Figure 4: Ex vivo detection of secreted and bioactive hlL-2 in supernatant of splenocytes from transgenic B6-hCD3EDG mice by ahCD3 VHH X NbALFA-LNPs containing hlL-2 encoded mRNA. Splenocytes of transgenic B6-hCD3EDG mice were transfected with respective LNPs (2000 ng RNA total, LNPs were formulated with an RNA mix of 1:1 w / w with or without ahCD3 VHH X NbALFA ligand). 50 IlJ / ml of recombinant hlL-2 was supplemented to medium as positive control, lx HBT buffer (10 mM HEPES, 10 w / v % Trehalose, pH 7.1) served as negative control. (A) Secreted hlL-2 concentration was analyzed by singleplex assay (hlL-2 MSD), hlL-2 concentration was calculated in pg / ml. (B) Bioactive hlL-2 was analyzed by HEK-Blue reporter assay, Absorbance was measured at OD 620-655 nm. n=l biological replicate / group.

[0198] Figure 5: hlL-2 mRNA containing LNPs lead to hlL-2 serum detection at 48 h. B6-hCD3EDG mice (n=5 / group) were injected i.v. with 20 pg of respective LNPs (LNPs were formulated with an RNA mix of 1:1 w / w with or without ahCD3 VHH X NbALFA ligand), lx HBT buffer (10 mM HEPES, 10 w / v % Trehalose, pH 7.1) served as negative control. Calculated serum hlL-2 concentration analyzed by singleplex assay (hlL-2 MSD). Two representative mice are shown.

[0199] Figure 6: T cell-restricted activation and proliferation by ahCD3 VHH X NbALFA-LNPs containing hlL-2 encoded mRNA in blood at 48 h. B6-hCD3EDG mice (n=5 / group) were injected i.v. with 20 pg of respective LNPs (LNPs were formulated with an RNA mix of 1:1 w / w with or without ahCD3 VHH X NbALFA ligand). 2 mg of BrdU were injected i.p. at 24 h. % BrdU+ lymphocyte sub-populations analyzed by flow cytometry (top). %BrdU+CD25+ lymphocyte sub-populations analyzed by flow cytometry (bottom). Data are presented as mean ± S.D., analyzed by a two-way ANOVA with Sidak's multiple comparison test, ns p > 0.5, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Three representative mice are shown.

[0200] Figure 7: Lymphocyte cell count sustained high in blood at 96 h after treatment with hlL-2 mRNA containing ahCD3 VHH X NbALFA-LNP. B6-hCD3EDG mice (n=5 / group) were injected i.v. with 20 pg of respective LNPs (LNPs were formulated with an RNA mix of 1:1 w / w with or without ahCD3 VHH X NbALFA ligand). Count of lymphocyte sub-populations analyzed by flow cytometry. Data are presented as mean ± S.D., analyzed by a two-way ANOVA with Sidak's multiple comparison test, *P < 0.1, **P < 0.01, ***P < 0.001, ****p < 0.0001.

[0201] Figure 8: Secretion of hlL-2 and hlFNy is related to ahCD3 VHH X NbALFA-LNP. Human PBMCs were transfected with respective LNPs at a dose of 1000 ng mRNA (LNPs were formulated with Luc:Thyl.l mRNA or hlL:2:Thyl.l mRNA, mRNA mix of 1:1 w / w and functionalized with or without ahCD3 VHH X NbALFA ligand. Supplemented recombinant hlL-2 (50 lU / ml) served as positive control, lx HBT buffer (10 mM HEPES, 10 w / v % Trehalose, pH 7.1) served as negative control. hlL-2 and h I F Ny secretion in supernatant was analyzed by Lumit™ immunoassay at 24 h after transfection. Data are presented as mean ± S.D., analyzed by an unpaired two-tailed t test, ns p > 0.5, **p < 0.01. n=3 technical replicates / group.

[0202] Figure 9: T cell-directed expression of reporter mRNA after ahCD3 VHH X NbALFA ligand functionalized LNPs after i.v. administration in blood at 48 h. B6-hCD3EDG mice (n=5 / group) were injected i.v. with 20 pg mRNA of respective LNPs (LNPs were formulated with Luc:Thyl.l mRNA or hlL:2:Thyl.l mRNA mix of 1:1 w / w and functionalized with or without ahCD3 VHH X NbALFA ligand). Transfection efficiency was analyzed by flow cytometry and is shown as %Thyl.l+ cells. Three representative mice are shown. Data are presented as mean ± S.D., analyzed by a one-way ANOVA with Sidak's multiple comparison test, ns p > 0.5, *p < 0.5.

[0203] Figure 10: CD4+ T cell number in the white pulp and the periphery of spleen is elevated after treatment with hlL-2 mRNA containing ahCD3 VHH X NbALFA-LNP at 96 h. B6-hCD3EDG mice (n=5 / group) were injected i.v. with 20 pg mRNA of respective LNPs (LNPs were formulated with Luc:Thyl.l mRNA or hlL:2:Thyl.l mRNA mix of 1:1 w / w and functionalized with or without ahCD3 VHH X NbALFA ligand). CD4+ (right) and CD8+ (left) T cell count was investigated by Immunohistochemistry (IHC) staining of spleen tissue. Scale bar 50 pm. One representative mouse / group is shown.

[0204] Figure 11: ahCD3 VHH X NbALFA ligand and hlL-2 mRNA induce a weight-loss at 96 h. B6- hCD3EDG mice (n=5 / group) were injected i.v. with 20 pg mRNA of respective LNPs. LNPs were formulated with irr. mRNA -- Luc:Thyl.l mRNA or hlL-2 mRNA = hlL:2:Thyl.l mRNA mix of 1:1 w / w and functionalized with or without ahCD3 VHH X NbALFA ligand. Body weight in %weight relative to day 0. Dashed line indicates the threshold of critical weight loss. Data are presented as mean ± S.D., analyzed by a two-way ANOVA with Tukey's multiple comparison test, ns p > 0.5, **p < 0.01, ****p < 0.0001.

[0205] Figure 12: Serum panIFNa level after i.v. injection LNPs at 48 h is analyzed. B6-hCD3EDG mice (n=5 / group) were injected i.v. with 20 pg mRNA of respective LNPs. LNPs were formulated with Luc:Thyl.l mRNA or hlL:2:Thyl.l mRNA mix of 1:1 w / w and functionalized with or without ahCD3 VHH X NbALFA ligand, lx HBT buffer (10 mM HEPES, 10 w / v % Trehalose, pH 7.1) served as negative control. Calculated serum IFNa level was determined by mouse panIFNa ELISA. Data are presented as mean ± S.D.. Two representative mice are shown. LLOQ = Lower Limit of Quantification.

[0206] Figure 13: hl L-2 mRNA containing LNPs induce no IL-6 production, but IL-5 secretion 48 h. B6- hCD3EDG mice (n=5 / group) were injected i.v. with 20 pg mRNA of respective LNPs (LNPs were formulated with Luc:Thyl.l mRNA or hlL:2:Thyl.l mRNA mix of 1:1 w / w and functionalized with or without ahCD3 VHH X NbALFA ligand), lx HBT buffer (10 mM HEPES, 10 w / v % Trehalose, pH 7.1) served as negative control. Calculated mouse serum Thl / Th2 profile analyzed by Multiplex Immunoassay using Luminex 200. Two representative mice are shown. Numbers indicate values out of defined range.

[0207] Figure 14: Physicochemical characterization of PLX-based formulation. PLXs were formulated with an Thyl.l / hlL-2 mRNA mix of 1:1 w / w, with or without amCD3 f(ab')2 X PGA. Size (A) and PDI (B) were in the expected range, measured via dynamic light scattering. Successful RNA incorporation was verified via agarose gel electrophoresis (not shown). n=4 technical replicates / group.

[0208] Figure 15: Cre:hlL-2 mRNA containing amCD3 f(ab')2 X PGA-PLXs are analyzed by high-content live imaging ex vivo. Isolated T cells from splenocytes of B6-Ai9 Cre reporter mice were transfected with respective PLX at a dose of 250 ng mRNA per 50x103 cells. High-content live imaging was performed for 70 h. PLXs were formulated with Thyl.l:Luc mRNA, Thyl.l:Cre mRNA and Cre:hlL-2 mRNA, mRNA mix 1:3 w / w and functionalized with or without amCD3 f(ab')2 X PGA ligand at w / w* 1.5. Supplemented recombinant IL-2 (50 lU / ml) served as positive control. (A) Transfection efficiency as count tdTomato+ T cells relative to count total cells over time. (B) Cell count as total cell count relative to 0 h time point. (C) Representative high- content live imaging images of Cre recombinase mediated tdTomato expression at 22 h post transfection. Images were recorded using Confocal Quantitative Image Cytometer Cell Voyager CQ.1 (Yokogawa). Data are presented as mean ± S.D. and analyzed by CellPathfinder from Yokogawa. n=3 technical replicates / group.

[0209] Figure 16: T cells express tdTomato after transfection with Cre mRNA containing amCD3 f(ab')2 X PGA-PLXs revealed by immunofluorescence staining. Splenocytes of B6-Ai9 Cre reporter mice were transfected with respective PLX at a dose of 750 ng mRNA per lxlO6cells. PLXs were formulated with irr. mRNA = Thyl.l:Luc mRNA, Cre mRNA = Thyl.l:Cre mRNA and Cre:hlL-2 mRNA, mRNA mix 1:3 w / w and functionalized with or without amCD3 ligand at w / w* 1.5. tdTomato+ cells are shown after immunofluorescence staining (IF). Scale bar 5 pm One representative IF staining / group.

[0210] Figure 17: DODMA-based ahCD3 VHH X NbALFA-LNPs deliverThyl.l reporter mRNA in a T cell- directed manner in vitro. Human PBMCs were transfected with respective LNPs at a dose of 1000 ng mRNA per 1x106 cells. LNPs were formulated with Thyl.l. mRNA and functionalized with or without ahCD3 VHH X NbALFA ligand at w / w* 0.35, 0.5 and 1.16. lx HBT buffer (10 mM HEPES, 10 w / v % Trehalose, pH 7.1) served as negative control. Transfection efficiency was analyzed by flow cytometry 6 h post-transfection and is shown as %Thyl.l+ cells. Data are presented as mean ±S.D., analyzed by a one-way ANOVA with Sidak's multiple comparison test, ns p > 0.5, **p < 0.01, ***p < 0.001. n=2 for w / w*0.35, 0.5, n=3 for w / w * 1.16 biological replicates / group.

[0211] Figure 18: Decreased mCD3 expression in T cells and T cell-directed mRNA delivery in murine splenocytes after transfection with amCD3 f(ab')2 X PGA-PLXs ex vivo. Murine splenocytes were transfected with respective PLX at a dose of 1000 ng mRNA (A) and at a dose range from 250 ng to 1000 ng mRNA (B) per 1x106 cells. PLXs were formulated with Thyl.l mRNA and functionalized with or without amCD3 f(ab')2 X PGA ligand at w / w* 0.2, 0.5, 1.0 and 1.5 (A), and at w / w* 1.0 and 1.5 (B). lx HBT buffer (10 mM HEPES, 10 w / v % Trehalose, pH 7.1) served as negative control. mCD3 expression (A) and cell-specific transfection efficiency (B) were analyzed by flow cytometry 6 h post-transfection. Numbers indicate the percentage of mCD3 expressing T cells (A). n=3 biological replicates / group (A), n=2 biological replicates / group (B). Detailed description

[0212] Although the present disclosure is further described in more detail below, it is to be understood that this disclosure is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0213] In the following, the elements of the present disclosure will be described in more detail. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present disclosure to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0214] The practice of the present disclosure will employ, unless otherwise indicated, conventional chemistry, biochemistry, pharmaceutical, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field.

[0215] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be 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 those that do not materially affect the basic and novel characteristic(s) 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, at each occurrence in the present application, the term "comprising" may be replaced with the term "consisting essentially of" or "consisting of". Likewise, at each occurrence in the present application, the term "consisting essentially of" may be replaced with the term "consisting of".

[0216] The terms "a", "an" and "the" and similar references used in the context of describing the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context.

[0217] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context.

[0218] The use of any and all examples, or exemplary language (e.g., "such as"), provided herein is intended merely to better illustrate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0219] The term "optional" or "optionally" as used herein means that the subsequently described event, circumstance or condition may or may not occur, and that the description includes instances where said event, circumstance, or condition occurs and instances in which it does not occur.

[0220] Where used herein, "and / or" is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "X and / or Y" is to be taken as specific disclosure of each of (i) X, (ii) Y, and (iii) X and Y, just as if each is set out individually herein.

[0221] In the context of the present disclosure, the term "about" denotes an interval of accuracy that the person of ordinary skill will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value by ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, and for example ±0.01%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±10%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±5%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±4%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±3%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±2%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±1%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.9%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.8%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.7%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.6%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.5%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.4%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.3%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.2%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.1%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.05%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.01%. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect.

[0222] 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. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0223] 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, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. In the following, definitions and embodiments will be provided which apply to all aspects of the present disclosure. Terms which are defined in the following have the meanings as defined unless otherwise indicated. Any undefined terms have their art recognized meanings.

[0224] Terms such as "reduce" or "inhibit" as used herein means the ability to cause an overall decrease, for example, of about 5% or greater, about 10% or greater, about 15% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 40% or greater, about 50% or greater, or about 75% or greater, in the level. The term "inhibit" or similar phrases includes a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero.

[0225] Terms such as "enhance" as used herein means the ability to cause an overall increase, or enhancement, for example, by at least about 5% or greater, about 10% or greater, about 15% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 40% or greater, about 50% or greater, about 75% or greater, or about 100% or greater in the level. Electric charge is a physical property that causes a matter to experience a force when near other electrically charged matter. Electric charge comes in two types, called positive and negative. Charged objects whose charges have the same sign (+ / + or - / -) repel one another, and objects whose charges have different (opposite) signs (+ / -) attract.

[0226] The electric charge of a macroscopic object such as a particle is the sum of the electric charges of the object that make it up. Objects may have equal numbers of positive and negative charges, in which case their charges cancel out, yielding a net charge of zero, thus making the objects neutral. Objects can have more positive charges than negative charges, in which case their charges do not cancel out, so the objects are positively charged (cationic). Objects can have more negative charges than positive charges, in which case their charges do not cancel out, so the objects are negatively charged (anionic). Net charge is the charge on a whole object such as a compound or particle.

[0227] An ion having an overall net positive charge is a cation while an ion having an overall net negative charge is an anion.

[0228] Particles described herein can be formed by adjusting a positive to negative charge, depending on the (+ / -) charge ratio of the particle forming components (and optionally the RNA). The amount of the different components having different charges can be easily determined by one skilled in the art in view of a loading amount upon preparation of the particles.

[0229] In some embodiments, the ratio of positive to negative charges in particles suitable for use herein is such that they may have a global positive charge.

[0230] If reference is made herein to a charge such as a positive charge, negative charge or neutral charge or a positive compound, negative compound or neutral compound this generally means that the charge mentioned is present at a selected pH, such as a physiological pH.

[0231] "Physiological pH" as used herein refers to a pH of about 7.4. In some embodiments, physiological pH is from 7.3 to 7.5. In some embodiments, physiological pH is from 7.35 to 7.45. In some embodiments, physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.

[0232] As used in the present disclosure, "% w / v" refers to weight by volume percent, which is a unit of concentration measuring the amount of solute in grams (g) expressed as a percent of the total volume of solution in milliliters (mL).

[0233] As used in the present disclosure, "% by weight" refers to weight percent, which is a unit of concentration measuring the amount of a substance in grams (g) expressed as a percent of the total weight of the total composition in grams (g).

[0234] As used in the present 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.

[0235] As used in the present disclosure, "mol % of the total lipid" is defined as the ratio of the number of moles of one lipid component to the total number of moles of all lipids, multiplied by 100. In this context, in some embodiments, the term "total lipid" includes lipids and lipid- like material.

[0236] The term "ionic strength" refers to the mathematical relationship between the number of different kinds of ionic species in a particular solution and their respective charges. Thus, ionic strength I is represented mathematically by the formula: in which c is the molar concentration of a particular ionic species and z the absolute value of its charge. The sum 1 is taken over all the different kinds of ions (i) in solution.

[0237] According to the disclosure, the term "ionic strength" in some embodiments relates to the presence of monovalent ions. Regarding the presence of divalent ions, in particular divalent cations, their concentration or effective concentration (presence of free ions) due to the presence of chelating agents is, in some embodiments, sufficiently low so as to prevent degradation of a nucleic acid. In some embodiments, the concentration or effective concentration of divalent ions is below the catalytic level for hydrolysis of the phosphodiester bonds between nucleotides such as RNA nucleotides. In some embodiments, the concentration of free divalent ions is 20 pM or less. In some embodiments, there are no or essentially no free divalent ions.

[0238] "Osmolality" refers to the concentration of a particular solute expressed as the number of osmoles of solute per kilogram of solvent.

[0239] The term "lyophilizing" or "lyophilization" refers to the freeze-drying of a substance by freezing it and then reducing the surrounding pressure (e.g., below 15 Pa, such as below 10 Pa, below 5 Pa, or 1 Pa or less) to allow the frozen medium in the substance to sublimate directly from the solid phase to the gas phase. Thus, the terms "lyophilizing" and "freeze- drying" are used herein interchangeably.

[0240] The term "spray-drying" refers to spray-drying a substance by mixing (heated) gas with a fluid that is atomized (sprayed) within a vessel (spray dryer), where the solvent from the formed droplets evaporates, leading to a dry powder.

[0241] The term "reconstitute" relates to adding a solvent such as water to a dried product to return it to a liquid state such as its original liquid state.

[0242] The term "recombinant" in the context of the present disclosure means "made through genetic engineering". In some embodiments, a "recombinant object" in the context of the present disclosure is not occurring naturally.

[0243] 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 is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring. The term "found in nature" means "present in nature" and includes known objects as well as objects that have not yet been discovered and / or isolated from nature, but that may be discovered and / or isolated in the future from a natural source. As used herein, the terms "room temperature" and "ambient temperature" are used interchangeably herein and refer to temperatures from at least about 15°C, e.g., from about 15°C to about 35°C, from about 15°C to about 30°C, from about 15°C to about 25°C, or from about 17°C to about 22°C. Such temperatures will include 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C and 22°C.

[0244] The term "EDTA" refers to ethylenediaminetetraacetic acid disodium salt. All concentrations are given with respect to the EDTA disodium salt.

[0245] The term "cryoprotectant" relates to a substance that is added to a formulation in order to protect the active ingredients during the freezing stages.

[0246] The term "lyoprotectant" relates to a substance that is added to a formulation in order to protect the active ingredients during the drying stages.

[0247] According to the present disclosure, the term "peptide" refers to substances which comprise about two or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100 or about 150, consecutive amino acids linked to one another via peptide bonds. The term "polypeptide" refers to large peptides, in particular peptides having at least about 151 amino acids. "Peptides" and "polypeptides" are both protein molecules. Thus, the terms "peptide", "protein" and "polypeptide" are used herein usually as synonyms.

[0248] Peptides and polypeptides disclosed herein may comprise a linear or a cyclized peptide sequence.

[0249] In some embodiments, the peptides disclosed herein comprises at least one cyclic portion, i.e., a polypeptide chain that contains a circular sequence of bonds that is referred to herein as a "cyclic peptide." The circular sequence can occur through a connection between the amino and carboxyl ends of the peptide; a connection between the amino end and a side chain; a connection between the carboxyl end and a side chain; or a connection between two side chains including sulfur groups of two cysteine amino acids by forming a disulfide bond, or more complicated arrangements.

[0250] 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 a mixture thereof. In some embodiments, the peptides and polypeptides disclosed herein comprise amino acid mimetics and amino acid analogs. In some embodiments, the peptides and polypeptides disclosed herein comprise non-naturally occurring amino acid sequences that are resistant to enzymatic cleavage.

[0251] 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 an isostere of the original residue.

[0252] 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, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-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. "Standard amino acid" refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides and polypeptides. "Nonstandard amino acid" refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and / or amino-terminal amino acid in a peptide or polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a peptide or polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a peptide or polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from 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 natural amino acids and their abbreviations:

[0253] Generally, amino acids are L-amino acids while D-amino acids are denoted by the prefix "D".

[0254] The prefix "homo" or "h" designates an a-amino acid that is otherwise similar to one of the common ones, but that contains one more methylene group in the carbon chain.

[0255] 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. It may also be possible to include non-peptide linkages and other chemical modification. For example, part or all of the peptide or polypeptide may be synthesized as a peptidomimetic, e.g., a peptoid (see, e.g., Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89:9367-71 and Horwell (1995) Trends Biotechnol.13:132-4). A peptide or polypeptide may include one or more (e.g., all) non-hydrolyzable bonds. Many non-hydrolyzable peptide bonds are known in the art, along with procedures for synthesis of peptides containing such bonds. Exemplary non- hydrolyzable bonds include -[CH2NH]- reduced amide peptide bonds, -[COCH2]- ketomethylene peptide bonds, -[CH(CN)NH]- (cyanomethylene)amino peptide bonds, - [CH2CH(OH)]- hydroxyethylene peptide bonds, -[CH2O]- oxymethylene peptide bonds, and - [CH2S]- thiomethylene peptide bonds (see e.g., U.S. Pat. No. 6,172,043).

[0256] The term "amide" as used herein, represents a group of formula "-NHC(O)-".

[0257] The term "thioamide" represents a group of formula "-NHC(S)-".

[0258] As used herein the term "disulfide bond", "disulfide bridge" or "disulfide" includes the covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group.

[0259] The term "ether" refers to a group or compound having an oxygen between two carbon atoms.

[0260] The term "thioether" refers to a group or compound having a sulfur between two carbon atoms.

[0261] The term "ester" refers a compound derived from an carboxylic acid and an alcohol by linking with formal loss of water the hydroxyl group of the -C(=O)OH group in the former and a hydroxy group of the latter. Thus, the term refers to the group -C(O)O-.

[0262] The term "thioester" refers to the group -C(O)S-.

[0263] The term "triazole" refers to chemical compounds that incorporate in their structure any heterocyclic structure having a five-membered ring of two carbon atoms and three nitrogen atoms (e.g., 1,2,3-triazole).

[0264] The term "portion" refers to a fraction. With respect to a particular structure such as an amino acid sequence or protein the term "portion" thereof may designate a continuous or a discontinuous fraction of said structure. The terms "part" and "fragment" are used interchangeably herein and refer to a continuous element. For example, a part of a structure such as an amino acid sequence or protein refers to a continuous element of said structure. When used in context of a composition, the term "part" means a portion of the composition. For example, a part of a composition may be any portion from 0.1% to 99.9% (such as 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%) of said composition.

[0265] "Fragment", with reference to an amino acid sequence (peptide or polypeptide), relates to a part of an amino acid sequence, i.e., a sequence which represents the amino acid sequence shortened at the N-terminus and / or C-terminus. A fragment shortened at the C-terminus (N- terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 3'-end of the open reading frame. A fragment shortened at the N-terminus (C- terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 5'-end of the open reading frame, as long as the truncated open reading frame comprises a start codon that serves to initiate translation. A fragment of an amino acid sequence comprises, e.g., at least 50 %, at least 60 %, at least 70 %, at least 80%, at least 90% of the amino acid residues from an amino acid sequence. A fragment of an amino acid sequence comprises, e.g., at least 6, in particular at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from an amino acid sequence. A fragment of an amino acid sequence comprises, e.g., a sequence of up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55, consecutive amino acids of the amino acid sequence.

[0266] "Variant," as used herein and with reference to an amino acid sequence (peptide or polypeptide), is meant an amino acid sequence that differs from a parent amino acid sequence by virtue of at least one amino acid (e.g., a different amino acid, or a modification of the same amino acid). The parent amino acid sequence may be a naturally occurring or wild type (WT) amino acid sequence, or may be a modified version of a wild type amino acid sequence. In some embodiments, the variant amino acid sequence has at least one amino acid difference as compared to the parent amino acid sequence, e.g., from 1 to about 20 amino acid differences, such as from 1 to about 10 or from 1 to about 5 amino acid differences compared to the parent. By "wild type" or "WT" or "native" as used herein and with reference to an amino acid sequence (peptide or polypeptide) is meant an amino acid sequence that is found in nature, including allelic variations. A wild type amino acid sequence, peptide or polypeptide has an amino acid sequence that has not been intentionally modified.

[0267] For the purposes of the present disclosure, "variants" of an amino acid sequence (peptide or polypeptide) may comprise 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, conformations, isoforms, allelic variants, species variants, and species homologs, in particular those which are naturally occurring. The term "variant" includes, in particular, fragments of an amino acid sequence. Amino acid insertion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible. Amino acid addition variants comprise amino- and / or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions may be in any position of the protein. Amino acid deletion variants that comprise the deletion at the N-terminal and / or C-terminal end of the protein are also called N-terminal and / or C- terminal truncation variants. Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Preference is given to the modifications being in positions in the amino acid sequence which are not conserved between homologous peptides or polypeptides and / or to replacing amino acids with other ones having similar properties. 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. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. 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.

[0268] In some embodiments, the degree of similarity, such as identity between a given amino acid sequence and an amino acid sequence which is a variant of said given amino acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the degree of similarity or identity is given for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is given, e.g., for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments continuous amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. The alignment for determining sequence similarity, such as sequence identity can be done with art known tools, such as using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0269] "Sequence similarity" indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.

[0270] The terms "% identical" and "% identity" or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or "window of comparison", in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of algorithms, e.g., the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website (e.g., at blast. ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC =align2seq). In some embodiments, the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match / Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used. In some embodiments, the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment.

[0271] Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.

[0272] In some embodiments, the degree of similarity or identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence. 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 continuous nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.

[0273] Homologous amino acid sequences exhibit according to the disclosure at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and, e.g., at least 95%, at least 98 or at least 99% identity of the amino acid residues.

[0274] The amino acid sequence variants described herein may readily be prepared by the skilled person, for example, by recombinant DNA manipulation. The manipulation of DNA sequences for preparing peptides or polypeptides having substitutions, additions, insertions or deletions, is described in detail in Molecular Cloning: A Laboratory Manual, 4th Edition, M.R. Green and J. Sambrook et al. (1989), eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012, for example. Furthermore, the peptides, polypeptides and amino acid variants described herein may be readily prepared with the aid of known peptide synthesis techniques such as, for example, by solid phase synthesis and similar methods.

[0275] 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 relates to any fragment or variant exhibiting one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., it is functionally equivalent. With respect to sequences of binding agents such as antibodies, one particular function is one or more binding activities displayed by the amino acid sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant", as used herein, in particular refers to a variant molecule or sequence that comprises an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and that is still capable of fulfilling one or more of the functions of the parent molecule or sequence, e.g., binding to a target molecule. In some embodiments, the modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present, e.g., function of the functional fragment or functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the parent molecule or sequence. However, in other embodiments, function of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0276] An amino acid sequence (peptide or polypeptide) "derived from" a designated amino acid sequence (peptide or polypeptide) refers to the origin of the first amino acid sequence. In some embodiments, the amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof. Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof. For example, it will be understood by one of ordinary skill in the art that the sequences suitable for use herein may be altered such that they vary in sequence from the naturally occurring or native sequences from which they were derived, while retaining the desirable activity of the native sequences.

[0277] In some embodiments, "isolated" means removed (e.g., purified) from the natural state or from an artificial composition, such as a composition from a production process. For example, a nucleic acid, peptide or polypeptide naturally present in a living animal is not "isolated", but the same nucleic acid, peptide or polypeptide partially or completely separated from the coexisting materials of its natural state is "isolated". An isolated nucleic acid, peptide or polypeptide can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0278] The term "bind" or "binding" relates to the non-covalent interaction with a target. In some embodiments, the term "bind" or "binding" relates to a specific binding. By the term "specific binding" or "specifically binds", as used herein, is meant a molecule such as an antibody or antigen receptor which recognizes a specific target molecule, but does not substantially recognize or bind other molecules in a sample or in a subject. 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. But, such cross-species reactivity does not itself alter the classification of an 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 itself alter the classification of an antibody as specific.

[0279] In some instances, the terms "specific binding" or "specifically binds", can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled "A" and the antibody, will reduce the amount of labeled A bound to the antibody.

[0280] As used herein, the terms "binding" or "capable of binding" typically is a binding with an affinity corresponding to a KD of about 107M or less, such as about 10'8M or less, such as about 109M or less, about 1010M or less, or about 1011M or even less, when determined using Bio-Layer Interferometry (BLI), or, for instance, when determined using surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument. In some embodiments, a binding moiety or agent binds to a predetermined target with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100-fold lower, for instance at least 1,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its affinity for binding to a non-specific target (e.g., BSA, casein).

[0281] The term "kd" (secx), as used herein, refers to the dissociation rate constant of a particular interaction, e.g., antibody-antigen interaction. Said value is also referred to as the kOff value.

[0282] The term "KD" (M), as used herein, refers to the dissociation equilibrium constant of a particular interaction, e.g., antibody-antigen interaction.

[0283] Generally, the terms "bind" or "binding" and "target" or "targeting" are used interchangeably herein.

[0284] The term "genetic modification" or simply "modification" includes the transfection of cells with nucleic acid. The term "transfection" relates to the introduction of nucleic acids, e.g., DNA and / or RNA, into a cell. For purposes of the present disclosure, the term "transfection" also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such cell, wherein the cell may be present in a subject, e.g., a patient, or the cell may be in vitro, e.g., outside of a patient. Thus, according to the present disclosure, a cell for transfection of a nucleic acid described herein can be present in vitro or in vivo, e.g. the cell can form part of an organ, a tissue and / or the body of a patient. According to the disclosure, transfection can be transient or stable. For some applications of transfection, it is sufficient if the transfected genetic material is only transiently expressed. RNA can be transfected into cells to transiently express its coded protein. Since the nucleic acid introduced in the transfection process is usually not integrated into the nuclear genome, the foreign nucleic acid will be diluted through mitosis or degraded. Cells allowing episomal amplification of nucleic acids greatly reduce the rate of dilution. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, a stable transfection must occur. Such stable transfection can be achieved by using virus-based systems or transposon-based systems for transfection, for example. Generally, cells that are genetically modified to express an antigen receptor are stably transfected with nucleic acid encoding the antigen receptor. RNA can be transfected into cells to transiently express its coded protein.

[0285] As used herein, the terms "linked", "fused", or "fusion" are used interchangeably. These terms refer to the joining together of two or more elements or components or domains.

[0286] 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 the two or more subunits. The translational fusion may be generated by genetically engineering the coding nucleotide sequence for one subunit in a reading frame with the coding nucleotide sequence of a further subunit. Subunits may be interspersed by a linker.

[0287] As used herein "endogenous" refers to any material from or produced inside an organism, cell, tissue or system.

[0288] As used herein, the term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0289] The term "autologous" is used to describe anything that is derived from the same subject. For example, "autologous transplant" refers to a transplant of tissue or organs derived from the same subject. Such procedures are advantageous because they overcome the immunological barrier which otherwise results in rejection.

[0290] The term "allogeneic" is used to describe anything that is derived from different individuals of the same species. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical.

[0291] The term "syngeneic" is used to describe anything that is derived from individuals or tissues having identical genotypes, i.e., identical twins or animals of the same inbred strain, or their tissues.

[0292] The term "heterologous" is used to describe something consisting of multiple different elements. As an example, the transfer of one individual's bone marrow into a different individual constitutes a heterologous transplant. A heterologous gene is a gene derived from a source other than the subject.

[0293] According to various embodiments of the present disclosure, a nucleic acid encoding a peptide or polypeptide is taken up by or introduced, i.e. transfected or transduced, into a cell which cell may be present in vitro or in a subject, resulting in expression of said peptide or polypeptide. The cell may, e.g., express the encoded peptide or polypeptide intracellularly (e.g. in the cytoplasm and / or in the nucleus), may secrete the encoded peptide or polypeptide, and / or may express it on the surface.

[0294] According to the present disclosure, terms such as "nucleic acid expressing" and "nucleic acid encoding" or similar terms are used interchangeably herein and with respect to a particular peptide or polypeptide mean that the nucleic acid, if present in the appropriate environment, e.g. within a cell, can be expressed to produce said peptide or polypeptide.

[0295] The term "expression" as used herein includes the transcription and / or translation of a particular nucleotide sequence.

[0296] In the context of the present disclosure, the term "transcription" relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA (especially mRNA). Subsequently, the RNA may be translated into peptide or polypeptide.

[0297] With respect to RNA, the term "expression" or "translation" relates to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino acids to make a peptide or polypeptide.

[0298] A medical preparation, in particular kit, comprising, for example, particles described herein may comprise instructional material or instructions. As used herein, "instructional material" or "instructions" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the present disclosure. The instructional material of the kit of the present disclosure may, for example, be affixed to a container which contains the compositions / formulations of the present disclosure or be shipped together with a container which contains the compositions / formulations. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compositions be used cooperatively by the recipient.

[0299] The term "average diameter" refers to the mean hydrodynamic diameter of particles as measured by dynamic light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Zaverage with the dimension of a length, and the polydispersity index (PDI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here "average diameter", "diameter" or "size" for particles is used synonymously with this value of the Zaverage-

[0300] In some embodiments, the "polydispersity index" is calculated based on dynamic light scattering measurements by the so-called cumulant analysis as mentioned in the definition of the "average diameter". Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of nanoparticles.

[0301] The "radius of gyration" (abbreviated herein as Rg) of a particle about an axis of rotation is the radial distance of a point from the axis of rotation at which, if the whole mass of the particle is assumed to be concentrated, its moment of inertia about the given axis would be the same as with its actual distribution of mass. Mathematically, Rgis the root mean square distance of the particle's components from either its center of mass or a given axis. For example, for a macromolecule composed of n mass elements, of masses m, ( / = 1, 2, 3, ..., n), located at fixed distances s, from the center of mass, Rgis the square-root of the mass average of s;2over all mass elements and can be calculated as follows:

[0302] The radius of gyration can be determined or calculated experimentally, e.g., by using light scattering. In particular, for small scattering vectors q the structure function S is defined as follows: wherein N is the number of components (Guinier's law).

[0303] The "hydrodynamic radius" (which is sometimes called "Stokes radius" or "Stokes-Einstein radius") of a particle is the radius of a hypothetical hard sphere that diffuses at the same rate as said particle. The hydrodynamic radius is related to the mobility of the particle, taking into account not only size but also solvent effects. For example, a smaller charged particle with stronger hydration may have a greater hydrodynamic radius than a larger charged particle with weaker hydration. This is because the smaller particle drags a greater number of water molecules with it as it moves through the solution. Since the actual dimensions of the particle in a solvent are not directly measurable, the hydrodynamic radius may be defined by the

[0304] Stokes-Einstein equation: wherein ke is the Boltzmann constant; 7" is the temperature; q is the viscosity of the solvent; and D is the diffusion coefficient. The diffusion coefficient can be determined experimentally, e.g., by using dynamic light scattering (DLS). Thus, one procedure to determine the hydrodynamic radius of a particle or a population of particles (such as the hydrodynamic radius of particles contained in a sample or control composition as disclosed herein or the hydrodynamic radius of a particle peak obtained from subjecting such a sample or control composition to field-flow fractionation) is to measure the DLS signal of said particle or population of particles (such as DLS signal of particles contained in a sample or control composition as disclosed herein or the DLS signal of a particle peak obtained from subjecting such a sample or control composition to field-flow fractionation).

[0305] The expression "light scattering" as used herein refers to the physical process where light is forced to deviate from a straight trajectory by one or more paths due to localized non- uniformities in the medium through which the light passes.

[0306] The term "UV" means ultraviolet and designates a band of the electromagnetic spectrum with a wavelength from 10 nm to 400 nm, i.e., shorter than that of visible light but longer than X- rays.

[0307] The expression "multi-angle light scattering" or "MALS" as used herein relates to a technique for measuring the light scattered by a sample into a plurality of angles. "Multi-angle" means in this respect that scattered light can be detected at different discrete angles as measured, for example, by a single detector moved over a range including the specific angles selected or an array of detectors fixed at specific angular locations. 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 comprising particles and by using an appropriate formalism (e.g., Zimm plot, Berry plot, or Debye plot), it is possible to determine the radius of gyration (Rg) and, thus, the size of said particles. Preferably, the Zimm plot is a graphical presentation using the following equation: wherein c is the mass concentration of the particles in the solvent (g / mL); A2 is the second virial coefficient (mol-mL / g2); P(d) is a form factor relating to the dependence of scattered light intensity on angle; R$ is the excess Rayleigh ratio (cm4); and K* is an optical constant that is equal to 4n2n0(dn / dc)2^'4^'1, where r|0is the refractive index of the solvent at the incident radiation (vacuum) wavelength, Ao is the incident radiation (vacuum) wavelength (nm), A / A is Avogadro's number (mol *), and dn / dc is the differential refractive index increment (mL / g) (cf., e.g., Buchholz et al. (Electrophoresis 22 (2001), 4118-4128); B.H. Zimm (J. Chem. Phys. 13 (1945), 141; P. Debye (J. Appl. Phys. 15 (1944): 338; and W. Burchard (Anal. Chem. 75 (2003), 4279-4291). Preferably, the Berry plot is calculated using the following term or the reciprocal thereof: wherein c, R& and K* are as defined above. Preferably, the Debye plot is calculated using the following term or the reciprocal thereof: wherein c, R$ and K* are as defined above.

[0308] The expression "dynamic light scattering" or "DLS" as used herein refers to a technique to determine the size and size distribution profile of particles, in particular with respect to the hydrodynamic radius of the particles. A monochromatic light source, usually a laser, is shot through a polarizer and into a sample. The scattered light then goes through a second polarizer where it is detected and the resulting image is projected onto a screen. The particles in the solution are being hit with the light and diffract the light in all directions. The diffracted light from the particles can either interfere constructively (light regions) or destructively (dark regions). This process is repeated at short time intervals and the resulting set of speckle patterns are analyzed by an autocorrelator that compares the intensity of light at each spot over time.

[0309] The expression "static light scattering" or "SLS" as used herein refers to a technique to determine the size and size distribution profile of particles, in particular with respect to the radius of gyration of the particles, and / or the molar mass of particles. A high-intensity monochromatic light, usually a laser, is launched in a solution containing the particles. One or many detectors are used to measure the scattering intensity at one or many angles. The angular dependence is needed to obtain accurate measurements of both molar mass and size for all macromolecules of radius. Hence simultaneous measurements at several angles relative to the direction of incident light, known as multi-angle light scattering (MALS) or multi-angle laser light scattering (MALLS), is generally regarded as the standard implementation of static light scattering.

[0310] Targeting compound

[0311] The particles described herein comprising an RNA payload, i.e., RNA encoding a polypeptide comprising a cytokine or a functional variant thereof, to be delivered comprise a moiety incorporating into the particle, e.g., a hydrophobic moiety (e.g., lipid), having a binding moiety covalently attached thereto. This moiety incorporating into the particle having a binding moiety covalently attached thereto is also referred to herein as "targeting compound". The moiety incorporating into the particle of the targeting compound relates to the part of the targeting compound that integrates into the particle comprising an RNA payload. The binding moiety of the targeting compound relates to the part of the targeting compound that binds to target immune cells or forms the binding partner for a docking compound which binds to target immune cells. Generally, the targeting compound is non-covalently incorporated into the particle comprising a payload, i.e., it forms an integral part of the particle, and the binding moiety of the targeting compound is covalently attached to a moiety incorporating into the particle in a manner such that it is available for binding to target immune cells or a docking compound.

[0312] In some embodiments, the binding moiety of the targeting compound comprises a peptide or protein (e.g., an antibody or antibody fragment or a peptide tag).

[0313] In some embodiments, the binding moiety of the targeting compound comprises a peptide or protein (e.g., an antibody or antibody fragment or a peptide tag) and is chemically linked, e.g., through a linker, to the moiety incorporating into the particle, e.g., hydrophobic moiety (e.g., lipid).

[0314] The targeting compound used herein comprises a moiety incorporating the targeting compound into the particle which allows it to be anchored in the particle.

[0315] In some embodiments, the targeting compound described herein comprises a hydrophobic component (e.g., lipid component) which allows it to be anchored in the particle. In some embodiments, the hydrophobic component comprises a moiety selected from vitamin E, dialkylamine, e.g., dimyristylamine (DMA), diacylglyceride, e.g., 1,2-dimyristoyl-sn-glycerol (DMG) and ceramide. In some embodiments, the hydrophobic moiety comprises two C8-C24 hydrocarbon chains. In some embodiments, the hydrophobic moiety comprises two C10-C18 hydrocarbon chains.

[0316] In some embodiments, the targeting compound described herein has as a hydrophobic group (e.g., lipid) a phospholipid, e.g., a biodegradable phospholipid such as phosphatidylethanolamine. In some embodiments, the targeting compound described herein has as a hydrophobic group (e.g., lipid) a glycerophospholipid. 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, as a phospholipid, DSPE will be used for its qualities of stability in the particles described herein. Moreover, as hydrophobic group (e.g., lipid), a compound having at least one alkyl chain providing hydrophobic anchoring to a particle as described herein may be used.

[0317] In some embodiments, the moiety incorporating the targeting compound into the particle interacts with the particle, e.g., one or more particle forming components, through electrostatic interaction. In some embodiments, the moiety incorporating the targeting compound into the particle comprises a charged moiety, e.g., a charged polymer. In some embodiments, the targeting compound is incorporated into the particle through a charge in the moiety incorporating the targeting compound into the particle interacting with an opposite charge in the particle, e.g., particle forming components of the particle having an opposite charge, and / or a particle having a net opposite charge (e.g., considering all charges of the particle forming components or considering all charges of the particle forming components and the nucleic acid payload). In some embodiments, the targeting compound is incorporated into the particle through a negative charge in the moiety incorporating the targeting compound into the particle interacting with a positive charge of the particle.

[0318] In some embodiments, it is possible to adjust the surface charge of the particle by incorporation of the targeting compound. In some embodiments, the surface charge can be adjusted based on the amount and type of the targeting compound, preferably based on a charged moiety of the targeting compound. In embodiments, the type and / or length of the charged moiety of the targeting compound is used for adjusting the surface charge.

[0319] In some embodiments, a targeting compound is incorporated into a particle, e.g., comprising a cationic particle forming component such as a cationic polymer, through a negative charge in the moiety incorporating the targeting compound into the particle interacting with a positive charge of the particle. In some embodiments, a targeting compound comprises a moiety incorporating the targeting compound into the particle comprising an anionic polymer. An anionic polymer described herein can be linear or branched, and comprises one or more anionic moieties or groups. In some embodiments, an anionic polymer is a polyanionic polymer, e.g., a polymer having one or more anionic groups. In some embodiments, an anionic group is a -CO2 , a -OSO3; or a -OPO32group. In some embodiments, the anionic polymer is a homopolymer. In some embodiments, the anionic polymer is a heteropolymer.

[0320] In some embodiments, an anionic polymer is polyglutamic acid. In some embodiments, an anionic polymer is poly-L-glutamic acid. In some embodiments, an anionic polymer is poly aspartic acid. In some embodiments, an anionic polymer is poly-L-aspartic acid. In some embodiments, an anionic polymer is a polyphosphate.

[0321] In some embodiments, an anionic polymer is a homopolymer. In some embodiments, an anionic polymer is a homopolymer comprising about 10 to about 150 repeating monomeric units. In some embodiments, an anionic polymer is a homopolymer comprising about 10 to about 100 repeating monomeric units. In some embodiments, an anionic polymer is a homopolymer comprising about 20 to about 100 repeating monomeric units. In some embodiments, an anionic polymer is a homopolymer comprising about 20 to about 80 repeating monomeric units. In some embodiments, an anionic polymer is a homopolymer comprising about 50 repeating monomeric units. In some embodiments, an anionic polymer is a homopolymer comprising about 100 repeating monomeric units.

[0322] In some embodiments, an anionic polymer is a poly-L-glutamic acid homopolymer comprising about 10 to about 150 repeating units of glutamic acid. In some embodiments, an anionic polymer is a poly-L-glutamic acid homopolymer comprising about 10 to about 100 repeating units of glumatic acid. In some embodiments, an anionic polymer is a poly-L-glutamic acid homopolymer comprising about 20 to about 100 repeating units of glumatic acid. In some embodiments, an anionic polymer is a poly-L-glutamic acid homopolymer comprising about 20 to about 80 repeating units of glutamic acid. In some embodiments, an anionic polymer is a poly-L-glutamic acid homopolymer comprising about 50 repeating units of glutamic acid. In some embodiments, an anionic polymer is a poly-L-glutamic acid homopolymer comprising about 100 repeating units of glutamic acid.

[0323] In some embodiments, glutamic acid is polymerized through formation of peptide bonds involving the a-carboxy group.

[0324] In some embodiments, the targeting compound used herein comprises a moiety incorporating the targeting compound into the particle comprising a polymer having a net negative charge. In some embodiments, the moiety incorporating the targeting compound into the particle comprises a polymer comprising one or more ionizable carboxy groups. In some embodiments, the moiety incorporating the targeting compound into the particle comprises a polyglutamic acid moiety.

[0325] In some embodiments, the targeting compound comprises a polymer. In some embodiments, the moiety incorporating into the particle, e.g., hydrophobic moiety (e.g., lipid) of the targeting compound and the binding moiety of the targeting compound are connected through the polymer.

[0326] In some embodiments, the polymer is a hydrophilic polymer. In some embodiments, the targeting compound comprises an amphiphilic derivative of the polymer. In some embodiments, the amphiphilic derivative of a polymer comprises a hydrophobic component (e.g., lipid component) which allows it to be anchored in the particle and a hydrophilic component of the polymer facing the outside of said particle, conferring hydrophilic properties at the surface thereof. In some embodiments, the amphiphilic derivatives of a polymer is inserted into the particle via its hydrophobic end. Consequently, the polymer component faces the outside of said particle and forms a protective hydrophilic shell surrounding the particle.

[0327] In some embodiments, the polymer portion of the targeting compound contributes to conferring stealth properties on the particles. In some embodiments, the plasmatic half-life of the particles described herein is greater than 2 hours, e.g., between 3 and 10 hours. This characteristic advantageously allows the particles to accumulate at the target immune cells and to liberate therein their contents (payload) within reasonable amounts of time. The effectiveness of the targeted delivery described herein therefore increases as a result.

[0328] The term "stealth" is used herein to describe the ability of the particles described herein not to be detected and then sequestered and / or degraded, or to be hardly detected and then sequestered and / or degraded, and / or to be detected and then sequestered and / or degraded late, by the immune system of the host to which they are administered.

[0329] Macrophages constitute one of the most important components of the immune system and play a predominant role in eliminating foreign particles, including liposomes and other colloidal particles, from the blood circulation. At the molecular level, the clearance of particles takes place in two steps: opsonization by the depositing of serum proteins (or "opsonins") at the surface of the particles followed by recognition and capture of the opsonized particles by macrophages.

[0330] Modification of the surface of particles with chains of hydrophilic and flexible polymers, e.g., polymers of the polyethylene glycol) type, confers them a steric protection by preventing the opsonins reaching the surface of the particles.

[0331] In some embodiments, the polymer for use herein is selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) (including derivatives thereof).

[0332] In some embodiments, a polymer is designed to sterically stabilize a particle by forming a protective hydrophilic layer. In some embodiments, a polymer can reduce association of a particle with serum proteins and / or the resulting uptake by the reticuloendothelial system when such particles are administered in vivo.

[0333] In some embodiments, the PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In some embodiments, the PEG is unsubstituted. In some embodiments, the PEG is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy or aryl groups. In some embodiments, the PEG has a molecular weight of from about 130 to about 50,000, in another embodiment about 150 to about 30,000, in another embodiment about 150 to about 20,000, in another embodiment about 150 to about 15,000, in another embodiment about 150 to about 10,000, in another embodiment about 150 to about 6000, in another embodiment about 150 to about 5000, in another embodiment about 150 to about 4000, in another embodiment about 150 to about 3000, in another embodiment about 300 to about 3000, in another embodiment about 1000 to about 3000, and in still another embodiment about 1500 to about 2500.

[0334] In some embodiments, the PEG moiety of the targeting compoundhas a molecular weight of 1000 or more. In some embodiments, the PEG moiety of the targeting compound comprises 10 units or more of formula (O-Cfh-CHzJn- In some embodiments, the PEG comprises from 20 to 200 ethylene oxide units, such as about 45 ethylene oxide units.

[0335] In some embodiments, the PEG comprises "PEG2k", also termed "PEG 2000", which has an average molecular weight of about 2000 Daltons. In some embodiments, DSPE-PEG2000, DSPE-PEG3000 and DSPE-PEG5000 are used as the amphiphilic derivative of a polymer.

[0336] In some embodiments, a pSar comprises between 2 and 200 sarcosine units, such as between 5 and 100 sarcosine units, between 10 and 50 sarcosine units, between 15 and 40 sarcosine units, e.g., about 23 sarcosine units.

[0337] In some embodiments, a pSar comprises the structure of the following general formula: wherein s is the number of sarcosine units.

[0338] In some embodiments, the POX and / or POZ polymer comprises between 2 and 200, between 2 and 190, between 2 and 180, between 2 and 170, between 2 and 160, between 2 and 150, between 2 and 140, between 2 and 130, between 2 and 120, between 2 and 110, between 2 and 100, between 2 and 90, between 2 and 80, between 2 and 70, between 5 and 200, between 5 and 190, between 5 and 180, between 5 and 170, between 5 and 160, between 5 and 150, between 5 and 140, between 5 and 130, between 5 and 120, between 5 and 110, between 5 and 100, between 5 and 90, between 5 and 80, between 5 and 70, between 10 and 200, between 10 and 190, between 10 and 180, between 10 and 170, between 10 and 160, between 10 and 150, between 10 and 140, between 10 and 130, between 10 and 120, between 10 and 110, between 10 and 100, between 10 and 90, between 10 and 80, or between 10 and 70 POX and / or POZ repeating units.

[0339] In some embodiments, the POX and / or POZ polymer comprises the following general formula: wherein a is an integer between 1 and 2; Ru is alkyl, in particular C1-3 alkyl, such as methyl, ethyl, iso-propyl, or n-propyl, and is independently selected for each repeating unit; and m refers to the number of POX and / or POZ repeating units.

[0340] In some embodiments, the POX and / or POZ polymer is a polymer of POX and comprises repeating units of the following general formula:

[0341] In some embodiments, the POX and / or POZ polymer is a polymer of POZ and comprises repeating units of the following general formula:

[0342] In any of the above embodiments of formulas, m (i.e., the number of repeating units in the polymer) preferably is between 2 and 190, such as between 2 and 180, between 2 and 170, between 2 and 160, between 2 and 150, between 2 and 140, between 2 and 130, between 2 and 120, between 2 and 110, between 2 and 100, between 2 and 90, between 2 and 80, between 2 and 70, between 5 and 200, between 5 and 190, between 5 and 180, between 5 and 170, between 5 and 160, between 5 and 150, between 5 and 140, between 5 and 130, between 5 and 120, between 5 and 110, between 5 and 100, between 5 and 90, between 5 and 80, between 5 and 70, between 10 and 200, between 10 and 190, between 10 and 180, between 10 and 170, between 10 and 160, between 10 and 150, between 10 and 140, between 10 and 130, between 10 and 120, between 10 and 110, between 10 and 100, between 10 and 90, between 10 and 80, or between 10 and 70. In certain embodiments, m is 2 to 180, such as 4 to 160, 6 to 140, 8 to 120 or 10 to 100, e.g., 20 to 80, 30 to 70, or 40 to 50. In some embodiments, the POX and / or POZ polymer is a copolymer comprising repeating units of the following general formulas: wherein the number of repeating units shown on the left in the copolymer is 1 to 199; the number of repeating units of formula on the right in the copolymer is 1 to 199; and the sum of the number of repeating units of formula on the left and the number of repeating units of formula on the right in the copolymer is 2 to 200.

[0343] In some embodiments of the oxazolinylated and / or oxazinylated moiety, the number of repeating units of formula on the left in the copolymer is 1 to 179, such as 1 to 159, 1 to 139, 1 to 119 or 1 to 99; the number of repeating units of formula on the right in the copolymer is Ito 179, such as I to 159, I to 139, Ito 119 or I to 99; and the sum of the number of repeating units of formula on the left and the number of repeating units of formula on the right in the copolymer is 2 to 180, such as 4 to 160, 6 to 140, 8 to 120 or 10 to 100, e.g., 20 to 80, 30 to 70, or 40 to 50.

[0344] In some of the above embodiments, Rn at each occurrence (i.e., in each repeating unit) may be the same alkyl group (e.g., Rn may be methyl in each repeating unit). In some alternative embodiments, Rn in at least one repeating unit differs from Rn in another repeating unit (e.g., for at least one repeating unit Rn is one specific alkyl (such as ethyl), and for at least one different repeating unit Rn is a different specific alkyl (such as methyl)). For example, each Rn may be selected from two different alkyl groups (such as methyl and ethyl) and not all Rn are the same alkyl.

[0345] In any of the above embodiments, Rn preferably is methyl or ethyl, more preferably methyl. Thus, in some embodiments, each Rn is methyl or each Rn is ethyl. In some alternative embodiments, Rn is independently selected from methyl and ethyl for each repeating unit, wherein in at least one repeating unit Rn is methyl, and in at least one repeating unit Rn is ethyl.

[0346] 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.

[0347] In some embodiments, the polymer comprises the following general formula: wherein X2and X1taken together are optionally substituted amide, optionally substituted thioamide or ester;

[0348] Y is -CH2-, -(CH2)2-, or -(CH2)3-; z is 2 to 24; and n is 1 to 100.

[0349] In some embodiments,

[0350] (i) when X1is -C(O)- then X2is -NR1-;

[0351] (ii) when X1is -NR1- then X2is -C(O)-;

[0352] (iii) when X1is -C(S)- then X2is -NR1-;

[0353] (iv) when X1is -NR1- then X2is -C(S)-;

[0354] (v) when X1is -C(O)- then X2is -O-; or

[0355] (vi) when X1is -O- then X2is -C(O)-; wherein R1is hydrogen or Ci-s alkyl.

[0356] In some embodiments, X1is -C(O)- and X2is -NR1-, wherein R1is hydrogen or Ci-s alkyl. In some embodiments, X1is -C(O)- and X2is -NR1-, wherein R1is hydrogen or methyl. In some embodiments, X1is -C(O)- and X2is -NR1-, wherein R1is hydrogen.

[0357] In some embodiments, Y is -CH2- or -(CH2)2-. In some embodiments, Y is -CH2-.

[0358] In some embodiments, the polymer comprises the following general formula: wherein

[0359] R1is hydrogen or Ci-s alkyl; z is 2 to 24; and n is 1 to 100.

[0360] In some embodiments of the above formulas, z is 2 to 10. In some embodiments, z is 2 to 7.

[0361] In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.

[0362] In some embodiments, the polymer comprises the following general formula: wherein

[0363] R1is hydrogen or Ci-g alkyl; and n is I to 100.

[0364] In some embodiments of the above formulas, R1is hydrogen or methyl. In some embodiments, R1is hydrogen.

[0365] In some embodiments, the polymer comprises the following general formula: wherein n is 1 to 100.

[0366] In some embodiments of the above formulas, n is 5 to 50. In some embodiments, n is 5 to 25.

[0367] 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.

[0368] In some embodiments, the molar proportion of the targeting compound integrated into the particles is between 0.5 and 20 mol% of the lipid molecules making up the particle, preferably between 1 and 10 mol%.

[0369] In some embodiments, the targeting compound comprises the following general formula:

[0370] L-X1-P-X2-B wherein

[0371] P comprises a polymer;

[0372] L comprises a moiety incorporating the targeting compound into the particle, e.g., a hydrophobic moiety (e.g., lipid) attached to a first end of the polymer;

[0373] B comprises a binding moiety attached to a second end of the polymer;

[0374] XI is absent or a first linking moiety; and

[0375] X2 is absent or a second linking moiety. In some embodiments, XI comprises a carbonyl group. In some embodiments, L comprises a phosphatidylethanolamine which may be linked to P by an amide group.

[0376] 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 the binding moiety.

[0377] In some embodiments, L comprises a lipid as described above. In some embodiments, L comprises DSPE (distearoylphosphatidylethanolamine), DPPE

[0378] (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine) which may be linked to P by an amide group. In some embodiments, P comprises a polymer as described above. In some embodiments, P comprises a polymer which provides stealth property, extends circulation half-life and / or reduces non-specific protein binding or cell adhesion. In some embodiments, P comprises a polymer selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) (including derivatives thereof). In some embodiments, P comprises polyethyleneglycol (PEG); e.g., PEG as described above.

[0379] In some embodiments, L-Xl-P comprises an amphiphilic derivative of a polymer as described above. In some embodiments, the amphiphilic derivative of a polymer comprises a conjugate of disteroyl-glycero-phosphoethanolamine (DSPE) and a polymer, e.g., a polymer as described above. In some embodiments, the amphiphilic derivative of a polymer comprises a disteroyl- glycero-phosphoethanolamine-polyethyleneglycol-conjugate (DSPE-PEG).

[0380] In some embodiments, the targeting compound is obtainable by reacting the thiol or cysteine reactive group of a reagent comprising an amphiphilic derivative of a polymer, e.g., a PEG reagent comprising a hydrophobic moiety (e.g., lipid), with a thiol or cysteine group of a compound comprising the binding moiety.

[0381] In some embodiments, the thiol or cysteine reactive group comprises a maleimide group.

[0382] In some embodiments, the PEG reagent comprises DSPE-PEG-maleimide. In some embodiments, the compound comprising the binding moiety comprises the formula SH(CH2)nC(O)-B, wherein n ranges from 1 to 5 and B comprises the binding moiety. In some embodiments, n is 2. In some embodiments, the targeting compound comprises the reaction product of 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)] with a compound comprising the formula SH(CH2)nC(O)-B, wherein n ranges from 1 to 5 and B comprises the binding moiety. In some embodiments, n is 2.

[0383] In some embodiments, the targeting compound comprises the following general formula: L-X1-P-X2-B wherein L, XI, P and B are as described above and X2 comprises a thiosuccinimide moiety.

[0384] In some embodiments, the targeting compound comprises the following general formula: wherein B comprises the binding moiety.

[0385] In some embodiments of the above formulas, B comprises a moiety comprising the structure -N-peptide-C(O)-NH2.

[0386] In some embodiments, the targeting compound comprises the following general formula: wherein P, X2 and B are as described above and Ri and R2 independently comprise an alkyl moiety. In some embodiments, at least one, e.g., each alkyl moiety is straight or branched, preferably straight. In some embodiments, at least one, e.g., each alkyl moiety has at least 8 carbon atoms, e.g., 8 to 24 such as 10 to 18 carbon atoms. Preferably, at least one, e.g., each alkyl moiety is the alkyl moiety of a fatty acid alcohol, more preferably at least one, e.g., each alkyl moiety is the alkyl moiety of a fatty acid alcohol having at least 8 carbon atoms, e.g., 8 to 24 such as 10 to 18 carbon atoms. Examples of alkyl moieties include -(CH2)I?CH3 (stearyl), - (CH2)i5CH3 (palmityl), and -(CFhJisCHs (myristyl). In some embodiments, R1R2N- in the above formula is 1,2-dimyristylamine, wherein both alkyl groups are -(CPhJisCHs (myristyl).

[0387] 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 comprises the following general formula: wherein n is 5 to 50, e.g., 5 to 25, e.g., 7 to 14, e.g., 10 to 25, e.g., 14 to 17. In some embodiments, n is 8 or 14. In some embodiments, n is 14. In some embodiments, Ri and R? in the above formula are -(CFhJisCHs (myristyl) and the polymer P comprises the following general formula: wherein n is 14.

[0388] In some embodiments, the targeting compound comprises the following general formula: wherein P, X2 and B are as described above and each of Rti and Rt2 is independently H or methyl. In some embodiments, Rti and Rt2 are both methyl. In some embodiments, Rti is methyl, and Rtz is H. In some embodiments, Rti is H, and Rt2 is methyl. In some embodiments, Rti and Rt2 are both H.

[0389] In some embodiments, the targeting compound comprises the following general formula: wherein P, X2 and B are as described above.

[0390] In some embodiments, the polymer P in the above formulas comprises poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof. In some embodiments, the polymer P comprises the following general formula: wherein n is 5 to 50, e.g., 5 to 25, e.g., 7 to 14, e.g., 10 to 25, e.g., 14 to 17. In some embodiments, n is 8 or 14. In some embodiments, n is 8. In some embodiments, n is 14.

[0391] In some embodiments, the targeting compound comprises the following general formula: wherein XI, P, X2 and B are as described above and Ri and R2 independently comprise an acyl moiety. In some embodiments, at least one, e.g., each acyl moiety is straight or branched, preferably straight. In some embodiments, at least one, e.g., each acyl moiety has at least 8 carbon atoms, e.g., 8 to 24 such as 10 to 18 carbon atoms. Preferably, at least one, e.g., each acyl moiety is the acyl moiety of a fatty acid, more preferably at least one, e.g., each acyl moiety is the acyl moiety of a fatty acid having at least 8 carbon atoms, e.g., 8 to 24 such as 10 to 18 carbon atoms. Examples of acyl moieties include CH3(CH2)ieC(O)- (stearoyl), CH3(CH2)i4C(O)- (palmitoyl), and CH3(CH2)i2C(O)- (myristoyl). In some embodiments, both acyl groups are CH3(CH2)ieC(O)- (stearoyl). In some embodiments, both acyl groups are CH3(CH2)i2C(O)- (myristoyl). In some embodiments, XI is absent or comprises -HPO3-(CH2)n- NH-, wherein n is 1 to 5, e.g., 2.

[0392] 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 comprises the following general formula: wherein n is 5 to 50, e.g., 5 to 25, e.g., 7 to 14, e.g., 10 to 25, e.g., 14 to 17. In some embodiments, n is 8 or 14. In some embodiments, n is 8. In some embodiments, n is 14.

[0393] In some embodiments, the polymer P comprises a pSar. In some embodiments, the polymer P comprises the following general formula: wherein s is 2 to 200, e.g., 5 to 100, e.g., 10 to 50, e.g., 15 to 40. In some embodiments, s is 20 or 23.

[0394] In some embodiments, the targeting compound comprises the following general formula: wherein P, X2 and B are as described above and Ri and R2 independently comprise an acyl moiety. In some embodiments, at least one, e.g., each acyl moiety is straight or branched, preferably straight. In some embodiments, at least one, e.g., each acyl moiety has at least 8 carbon atoms, e.g., 8 to 24 such as 10 to 18 carbon atoms. Preferably, at least one, e.g., each acyl moiety is the acyl moiety of a fatty acid, more preferably at least one, e.g., each acyl moiety is the acyl moiety of a fatty acid having at least 8 carbon atoms, e.g., 8 to 24 such as 10 to 18 carbon atoms. Examples of acyl moieties include CH3(CH2)ieC(O)- (stearoyl), CH3(CH2)i4C(O)- (palmitoyl), and CH3(CH2)i2C(O)- (myristoyl). In some embodiments, both acyl groups are CH3(CH2)i6C(O)- (stearoyl). In some embodiments, both acyl groups are CH3(CH2)i2C(O)- (myristoyl).

[0395] 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 comprises the following general formula: wherein n is 5 to 50, e.g., 5 to 25, e.g., 7 to 14, e.g., 10 to 25, e.g., 14 to 17. In some embodiments, n is 8 or 14. In some embodiments, n is 8. In some embodiments, n is 14.

[0396] In some embodiments, n is 8 and Ri and R2 are CH3(CH2)ieC(O)- (stearoyl). In some embodiments, n is 14 and Ri and R3are CH3(CH2)ieC(O)- (stearoyl). In some embodiments, n is 8 and Ri and R2 are CH3(CH2)i2C(O)- (myristoyl). In some embodiments, n is 14 and Ri and R2are CH3(CH2)i2C(O)- (myristoyl).

[0397] In some embodiments, the polymer P comprises a pSar. In some embodiments, the polymer P comprises the following general formula: wherein s is 2 to 200, e.g., 5 to 100, e.g., 10 to 50, e.g., 15 to 40. In some embodiments, s is 20 or 23.

[0398] In some embodiments, s is 20 and Ri and R2 are CH3(CH2)i6C(O)- (stearoyl).

[0399] In some embodiments, s is 20 and Ri and R2 are CH3(CH2)i2C(O)- (myristoyl).

[0400] In some embodiments, X2 in the above formulas comprises the reaction product of a thiol or cysteine reactive group, e.g., a maleimide group, with a compound comprising a thiol or cysteine group. In some embodiments, the compound comprising a thiol or cysteine group comprises the formula SH(CH2)nC(O)-, wherein n ranges from 1 to 5. In some embodiments, n is 2. In some embodiments, X2 comprises a thiosuccinimide moiety.

[0401] In some embodiments, X2 comprises the following general formula: wherein nl and n2 are independently 1 to 5. In some embodiments, nl is 1 and n2 is 2. In some embodiments, nl is 2 and n2 is 1.

[0402] The present disclosure provides in one aspect, a targeting compound as described herein. In some embodiments of the targeting compound, the binding moiety comprises a moiety binding to a cell surface antigen, e.g., a primary targeting moiety described herein. In some embodiments of the targeting compound, the binding moiety comprises a moiety binding to a docking compound. In some embodiments of the targeting compound, the binding moiety comprises a tag such as an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein. Accordingly, the present disclosure provides in one aspect, a compound the following general formula:

[0403] L-X1-P-X2-B wherein

[0404] P comprises a polymer;

[0405] [.comprises a moiety incorporatingthe compound into the particle, e.g., a hydrophobic moiety (e.g., lipid) attached to a first end of the polymer;

[0406] B comprises a primary targeting moiety described herein, attached to a second end of the polymer;

[0407] XI is absent or a first linking moiety; and

[0408] X2 is absent or a second linking moiety.

[0409] The present disclosure further provides in one aspect, a compound the following general formula:

[0410] L-X1-P-X2-B wherein

[0411] P comprises a polymer;

[0412] L comprises a moiety incorporating the compound into the particle, e.g., a hydrophobic moiety (e.g., lipid) attached to a first end of the polymer;

[0413] B comprises a tag such as an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein, attached to a second end of the polymer;

[0414] XI is absent or a first linking moiety; and

[0415] X2 is absent or a second linking moiety.

[0416] In some embodiments, XI comprises a carbonyl group. In some embodiments, L comprises a phosphatidylethanolamine which may be linked to P by an amide group.

[0417] 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 the epitope tag. In some embodiments, X2 comprises a thiosuccinimide moiety. In some embodiments, L comprises a lipid as described above. In some embodiments, L comprises DSPE (distearoylphosphatidylethanolamine), DPPE

[0418] (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine) which may be linked to P by an amide group. In some embodiments, P comprises a polymer as described above. In some embodiments, P comprises a polymer which provides stealth property, extends circulation half-life and / or reduces non-specific protein binding or cell adhesion. In some embodiments, P comprises a polymer selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) (including derivatives thereof). In some embodiments, P comprises polyethyleneglycol (PEG); e.g., PEG as described above.

[0419] In some embodiments, L-Xl-P comprises an amphiphilic derivative of a polymer as described above. In some embodiments, the amphiphilic derivative of a polymer comprises a conjugate of disteroyl-glycero-phosphoethanolamine (DSPE) and a polymer, e.g., a polymer as described above. In some embodiments, the amphiphilic derivative of a polymer comprises a disteroyl- glycero-phosphoethanolamine-polyethyleneglycol-conjugate (DSPE-PEG).

[0420] In some embodiments, the targeting compound is obtainable by reacting the thiol or cysteine reactive group of a reagent comprising an amphiphilic derivative of a polymer, e.g., a PEG reagent comprising a hydrophobic moiety (e.g., lipid), with a thiol or cysteine group of a compound comprising the primary targeting moiety or epitope tag.

[0421] In some embodiments, the thiol or cysteine reactive group comprises a maleimide group.

[0422] In some embodiments, the PEG reagent comprises DSPE-PEG-maleimide. In some embodiments, the compound comprising the primary targeting moiety or epitope tag comprises the formula SH(CH2)nC(O)-B, wherein n ranges from 1 to 5 and B comprises the primary targeting moiety or epitope tag. In some embodiments, n is 2.

[0423] In some embodiments, the targeting compound comprises the reaction product of 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)] with a compound comprising the formula SH(CH2)nC(O)-B, wherein n ranges from 1 to 5 and B comprises the primary targeting moiety or epitope tag. In some embodiments, n is 2.

[0424] In some embodiments, the targeting compound comprises the following general formula: wherein B comprises an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein. In some embodiments, the targeting compound comprises the following general formula: wherein X2 is as described above, Ri and R2 are CH3(CH2)ieC(O)- (stearoyl) or CH3(CH2)i2C(O)- (myristoyl), polymer P comprises the following general formula: wherein n is 5 to 50, e.g., 5 to 25, e.g., 7 to 14, e.g., 10 to 25, e.g., 14 to 17, e.g., 8 or 14, and B comprises an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein.

[0425] In some embodiments, n is 8 and Ri and R2 are CH3(CH2)i6C(O)- (stearoyl). In some embodiments, n is 14 and Ri and R2 are CH3(CH2)i6C(O)- (stearoyl).

[0426] In some embodiments, n is 8 and Ri and R2 are CH3(CH2)i2C(O)- (myristoyl). In some embodiments, n is 14 and Ri and R2 are CHs(CH2)i2C(O)- (myristoyl).

[0427] In some embodiments, X2 comprises the following general formula:

[0428] In some embodiments, the targeting compound comprises the following general formula: wherein X2 is as described above, Ri and R2 are CH3<CH2)i6C(O)- (stearoyl) or CH3(CH2)i2C(O)- (myristoyl), polymer P comprises the following general formula: wherein s is 2 to 200, e.g., 5 to 100, e.g., 10 to 50, e.g., 15 to 40, e.g., 20 or 23, and B comprises an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein. In some embodiments, s is 20 and Ri and Fb are CH3(CH2)ieC(O)- (stearoyl).

[0429] In some embodiments, s is 20 and Ri and Rz are CHs(CH2)i2C(O)- (myristoyl).

[0430] In some embodiments, X2 comprises the following general formula:

[0431] In some embodiments, B comprises a moiety comprising the structure -N-peptide-C(O)-NH2, wherein peptide comprises an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein.

[0432] The present disclosure provides in one aspect, a targeting compound as described above which is integrated in a particle (e.g., a particle as described herein), e.g., via a hydrophobic component (e.g., lipid component) of the targeting compound.

[0433] Primary targeting moiety

[0434] According to the disclosure, an RNA payload is delivered specifically to a target immune cell by providing a moiety that binds to a target on target immune cells, e.g., an antigen on target immune cells, thus targeting particles comprising the RNA payload to the target immune cells. In some embodiments, the moiety that binds to a target on target immune cells is comprised by a compound (targeting compound) which is an integral part of a particle carrying the payload. In these embodiments, the targeting compound comprises a binding moiety that binds to target immune cells.

[0435] In some embodiments, the moiety that binds to a target on target immune cells is comprised by a compound (docking compound) further comprising a moiety that binds to a targeting compound which is an integral part of a particle carrying the payload and comprising a moiety for binding to the docking compound. In these embodiments, the targeting compound itself preferably does not comprise a moiety that binds to a target on target immune cells. Rather, the targeting compound comprises a binding moiety that forms the binding partner for a docking compound which binds to target immune cells.

[0436] The target on target immune cells is also referred to herein as "primary target". In some embodiments, a primary target is a cell surface antigen on target immune cells.

[0437] A "primary targeting moiety" as used herein relates to the part of the targeting compound or docking compound which binds to a primary target, e.g., a cell surface antigen on target immune cells. Such targeting moieties are typically moieties that have affinity for cell surface targets. These moieties can be any peptide or protein (e.g. antibodies or antibody fragments) binding to the primary target. Particular embodiments of suitable primary targeting moieties for use herein include cell surface antigen binding moieties, such as antibodies, antibody fragments and DARPins. Other examples of primary targeting moieties are peptides or proteins which bind to a receptor.

[0438] A primary targeting moiety preferably binds with high specificity and / or high affinity and the bond with the primary target is preferably stable within the body.

[0439] In order to allow specific targeting of primary targets, the primary targeting moiety of the targeting compound or docking compound can comprise compounds including but not limited to antibodies, antibody fragments, e.g. Fab2, Fab, scFV, VHH domains, and other proteins or peptides.

[0440] According to some embodiments, the primary target is a cell surface antigen such as a T cell antigen, e.g., CD3, such as CD3e, CD8 or CD4, and suitable primary targeting moieties include but are not limited to, peptides and polypeptides targeting the cell surface antigen, e.g., antibodies, antibody fragments and DARPins.

[0441] According to some embodiments, the primary target is a receptor and suitable primary targeting moieties include but are not limited to, the ligand of such a receptor or a part thereof which still binds to the receptor, e.g., a receptor binding peptide in the case of receptor binding protein ligands.

[0442] According to some embodiments, the primary target and primary targeting moiety are selected so as to result in the specific or increased targeting of certain cells. This can be achieved by selecting primary targets with cell-specific expression. For example, T cell antigens, e.g., those described herein, may be expressed in T cells while they are not expressed or expressed in a lower amount in other cells.

[0443] Docking compound

[0444] In some embodiments, a "docking compound" is used to form a connection between a primary target, e.g., a target immune cell or an antigen on target immune cells, and a targeting compound which is integrated into a particle comprising an RNA payload to be delivered to a target immune cell. In some embodiments, a connection between a primary target, e.g., a target immune cell or an antigen on target immune cells, and a docking compound is a non- covalent connection. In some embodiments, a connection between a docking compound and a targeting compound is a non-covalent or covalent connection. In some embodiments, the targeting compound comprises a binding moiety for binding to the docking compound which is covalently attached to a hydrophobic moiety (e.g., lipid). The hydrophobic moiety (e.g., lipid) forms part of said particle.

[0445] In some embodiments, a docking compound comprises a "primary targeting moiety", e.g., a moiety targeting a cell surface antigen on target immune cells, that is capable of binding to the primary target of interest, e.g., a cell surface antigen on target immune cells. In some embodiments, a "primary targeting moiety" as used herein relates to the part of the docking compound which binds to a primary target.

[0446] The docking compound further comprises a group which serves as a binding partner for a respective binding moiety of a targeting compound. The portion of the targeting compound comprising the moiety incorporating the targeting compound into the particle, e.g., the hydrophobic moiety (e.g., lipid) (having a binding moiety for the docking compound covalently attached) integrates into a particle carrying a payload and thus forms a connection between the particle and the docking compound. The moiety of the docking compound binding to the targeting compound and the primary targeting moiety are linked to each other, preferably by a covalent linkage.

[0447] According to some embodiments, the docking compound comprises a bispecific molecule, such as a bispecific polypeptide, e.g., a bispecific antibody. In some embodiments, the docking compound comprises a binding domain binding to a primary target and a binding domain binding to a targeting compound. In some embodiments, the docking compound comprises an antibody or antibody fragment binding to a primary target and an antibody or antibody fragment binding to a targeting compound. 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 binding to a 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 binding to a primary target comprises a single-domain antibody such as a VHH. In some embodiments, the binding domain binding to a targeting compound comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. In some embodiments, the binding domain binding to a targeting compound comprises a single-domain antibody such as a VHH.

[0448] In some embodiments, the docking compound comprises a fusion protein which comprises a binding domain binding to a primary target and a binding domain binding to a targeting compound.

[0449] In some embodiments, the docking compound comprises a single peptide chain. In some embodiments, the single peptide chain comprises a portion, e.g., antibody, antibody fragment or DARPin, binding to a primary target and a portion, e.g., antibody or antibody fragment, binding to a targeting compound. In some embodiments, the antibody fragments are VHH, scFv, or a mixture thereof. In different embodiments, the docking compound comprises one of the following structures (from N- to C-terminus):

[0450] VHH (a targeting compound)-optional linker-VHH (a primary target) VHH (a primary target)-optional linker-VHH (a targeting compound) VHH (a targeting compound)-optional linker-scFv (a primary target) scFv (a primary target)-optional linker-VHH (a targeting compound) VHH (a primary target)-optional linker-scFv (a targeting compound) scFv (a targeting compound)-optional linker-VHH (a primary target) scFv (a targeting compound)-optional linker-scFv (a primary target) scFv (a primary target)-optional linker-scFv (a targeting compound)

[0451] The present disclosure provides in one aspect, a docking compound as described herein. In some embodiments, the docking compound comprises a bispecific molecule, such as a bispecific polypeptide, e.g., a bispecific antibody, wherein one specificity binds to an epitope tag, e.g., an ALFA-tag and the other scpecificity binds to a primary target, e.g., a cell surface antigen on target immune cells. In some embodiments, the specificity which binds to an epitope tag is an antibody or antibody fragment such as an NbALFA-nanobody (NbALFA). In some embodiments, the specificity which binds to a primary target is an antibody, antibody fragment or DARPin. In some embodiments, the moiety targeting a primary target of the docking compound is selected from the group consisting of an anti-primary target DARPin, an anti-primary target VHH and an anti-primary target scFv and / or the moiety binding to a targeting compound of the docking compound is an NbALFA-nanobody (NbALFA). 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 antiprimary target scFv. In some embodiments, the primary target is a T cell antigen, e.g., CD3, such CD3e, CD4 or CD8. In some embodiments, the docking compound comprises a bispecific antibody comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti- CD3 VHH. In some embodiments, the docking compound comprises a bispecific antibody comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-CD3 scFv. In some embodiments, the docking compound comprises a bispecific molecule comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-CD3 DARPin. In some embodiments, the docking compound comprises a bispecific antibody comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-CD4 VHH. In some embodiments, the docking compound comprises a bispecific antibody comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-CD4 scFv. In some embodiments, the docking compound comprises a bispecific molecule comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-CD4 DARPin. In some embodiments, the docking compound comprises a bispecific antibody comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-CD8 VHH. In some embodiments, the docking compound comprises a bispecific antibody comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-CD8 scFv. In some embodiments, the docking compound comprises a bispecific molecule comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti- CD8 DARPin.

[0452] Interacting moieties on the targeting compound and on the docking compound

[0453] In some embodiments, the moiety on the targeting compound and the moiety on the docking compound interacting which each other non-covalently bind to each other.

[0454] In some embodiments, the moieties on the targeting compound and on the docking compound interacting which each other bind to each other under physiological conditions.

[0455] In some embodiments, the moieties on the targeting compound and on the docking compound interacting which each other are antibody / antigen systems.

[0456] In some embodiments, the moiety of the targeting compound binding to the docking compound comprises a peptide or protein, e.g., a peptide tag, and the moiety of the docking compound binding to the targeting compound comprises a binder, e.g., an antibody or antibody fragment, binding to the peptide or protein.

[0457] In some embodiments, the moiety of the docking compound binding to the targeting compound comprises a peptide or protein, e.g., a peptide tag, and the moiety of the targeting compound bindingto the docking compound comprises a binder, e.g., an antibody or antibody fragment, binding to the peptide or protein. In some embodiments, the moieties on the targeting compound and on the docking compound interacting which each other comprise an epitope tag / binder system.

[0458] The term "tag" as used herein refers to a chemical moiety which can be bound by another moiety.

[0459] As used herein, an "epitope tag" refers to a stretch of amino acids to which an antibody or proteinaceous molecule with antibody-like function can bind. In some embodiments, the epitope tag comprises an ALFA-tag. In some embodiments, the epitope tag / binder system comprises an ALFA-tag and an ALFA-specific single-domain antibody (sdAb), NbALFA-nanobody.

[0460] In some embodiments, an ALFA-tag comprises the amino acid sequence

[0461] -AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12-AA13-AA14-, wherein the amino acids of AAO, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13 and AA14 are: AAO is Pro or deleted;

[0462] AA1 is Ser, Gly, Thr, or Pro;

[0463] AA2 is Arg, Gly, Ala, Glu, or Pro;

[0464] AA3 is Leu, lie, or Vai;

[0465] AA4 is Glu or Gin;

[0466] AA5 is Glu or Gin;

[0467] AA6 is Glu or Gin;

[0468] AA7 is Leu, lie, or Vai;

[0469] AA8 is Arg, Ala, Gin, or Glu;

[0470] AA9 is Arg, Ala, Gin, or Glu;

[0471] AA10 is Arg;

[0472] AA11 is Leu;

[0473] AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;

[0474] AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and

[0475] AA14 is Pro or deleted.

[0476] In some embodiments, an ALFA-tag comprises a sequence selected from the group consisting of SRLEEELRRRLTE, PSRLEEELRRRLTE, SRLEEELRRRLTEP, and PSRLEEELRRRLTEP.

[0477] In some embodiments, an ALFA-tag comprises the cyclized amino acid sequence

[0478] -AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12-AA13-AA14-, wherein the side-chains of any two of the amino acids of AAO, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13 and AA14 (XI, X2) are connected covalently; and wherein the amino acids of AAO, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13 and AA14 which are not XI and X2 are: AAO is Pro or deleted;

[0479] AA1 is Ser, Gly, Thr, or Pro;

[0480] AA2 is Arg, Gly, Ala, Glu, or Pro;

[0481] AA3 is Leu, lie, or Vai;

[0482] AA4 is Glu or Gin;

[0483] AA5 is Glu or Gin;

[0484] AA6 is Glu or Gin;

[0485] AA7 is Leu, He, or Vai;

[0486] AA8 is Arg, Ala, Gin, or Glu;

[0487] AA9 is Arg, Ala, Gin, or Glu;

[0488] AA10 is Arg;

[0489] AA11 is Leu;

[0490] AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;

[0491] AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and

[0492] AA14 is Pro or deleted.

[0493] In some embodiments, XI and X2 are separated by 2 or 3 amino acids.

[0494] In some embodiments, AA5 is XI and AA9 is X2, AA5 is XI and AA8 is X2, AA9 is XI and AA13 is X2, AA6 is XI and AA9 is X2, AA9 is XI and AA12 is X2, AA10 is XI and AA13 is X2, AA6 is XI and AA10 is X2 or AA4 is XI and AA8 is X2.

[0495] In some embodiments, an ALFA-tag comprises a cyclized amino acid sequence selected from the group consisting of a. -AA0-AAl-AA2-AA3-AA4-cyclo(Xl-AA6-AA7-AA8-X2)-Arg-Leu-AA12-AA13-AA14-, b. -AA0-AAl-AA2-AA3-AA4-cyclo(Xl-AA6-AA7-X2)-AA9-Arg-Leu-AA12-AA13-AA14-, c. -AA0-AAl-AA2-AA3-AA4-AA5-AA6-AA7-AA8-cyclo(Xl-Arg-Leu-AA12-X2)-AA14-, d. -AA0-AAl-AA2-AA3-AA4-AA5-cyclo(Xl-AA7-AA8-X2)-Arg-Leu-AA12-AA13-AA14-, e. -AA0-AAl-AA2-AA3-AA4-AA5-AA6-AA7-AA8-cyclo(Xl-Arg-Leu-X2)-AA13-AA14-, f. -AA0-AAl-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-cyclo(Xl-Leu-AA12-X2)-AA14-, g. -AA0-AAl-AA2-AA3-AA4-AA5-cyclo(Xl-AA7-AA8-AA9-X2)-Leu-AA12-AA13-AA14-, and h. -AA0-AAl-AA2-AA3-cyclo(Xl-AA5-AA6-AA7-X2)-AA9-Arg-Leu-AA12-AA13-AA14-, wherein the side-chains of Xi and X2 amino acid residues are connected covalently; AAO is Pro or deleted;

[0496] AA1 is Ser, Gly, Thr, or Pro;

[0497] AA2 is Arg, Gly, Ala, Glu, or Pro;

[0498] AA3 is Leu, He, or Vai;

[0499] AA4 is Glu or Gin;

[0500] AA5 is Glu or Gin;

[0501] AA6 is Glu or Gin;

[0502] AA7 is Leu, He, or Vai;

[0503] AA8 is Arg, Ala, Gin, or Glu;

[0504] AA9 is Arg, Ala, Gin, or Glu;

[0505] AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;

[0506] AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and

[0507] AA14 is Pro or deleted.

[0508] In some embodiments, Xi and X2 in the peptides disclosed herein are connected covalently via an amide, disulfide, thioether, ether, ester, thioester, thioamide, alkylene, alkenylene, alkynylene, and / or 1,2,3-triazole.

[0509] In some embodiments, a cyclized amino acid sequence described herein is generated by linking an amino group of a side-chain of one of Xi and X2 to the carboxyl group of a side-chain of the other of Xi and X2 via an amide bond. The amino group of the side chain of an amino acid that possesses a pendant amine group, e.g., lysine or a lysine derivative, and the carboxyl group of the side chain of an acidic amino acid, e.g., aspartic acid, glutamic acid or a derivative thereof, can be used to generate a cyclized amino acid sequence via an amide bond.

[0510] In some embodiments, a cyclized amino acid sequence described herein is generated by linking a sulfhydryl group of a side-chain of one of Xi and X2 to the sulfhydryl group of a sidechain of the other of Xi and X2 via a disulfide bond. Sulfhydryl group-containing amino acids include cysteine and other sulfhydryl-containing amino acids as Pen.

[0511] In some embodiments, Xi 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 Xi is Glu, DGlu, Asp, or DAsp, X2 is Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, or DDap; when XI is Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, or DDap, X2 is Glu, DGlu, Asp, or DAsp; and when XI is Cys, DCys, hCys, DhCys, Pen, or DPen, X2 is Cys, DCys, hCys, DhCys, Pen, or DPen.

[0512] In some embodiments, Xi is Glu and X2 is Lys. In some embodiments, -cyclo(Glu - Lys)-, - c(G I u - Lys)-, -cyclo(E - K)-, -c(E - K)-, -E K- cyclo, or -cycloE-— cycloK- comprises the following structure:

[0513] In some embodiments, Xi is Lys and X2 is Glu. In some embodiments, -cyclo(Lys - Glu)-, - c(Lys - Glu)-, -cyclo(K - E)-, -c(K - E)-, -K - E- cyclo, or cycloK - cycloE- comprises the following structure:

[0514] In some embodiments, Xi 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- comprises the following structure:

[0515] Particular cyclized amino acid sequences of the above-identified generic formulas include, for example,

[0516] -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-,

[0517] -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-,

[0518] -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-cyclofGlu-Glu-Leu-Arg-LysJ-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DGIu-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-DCys)-Arg-Arg-Leu-Thr-Glu-, -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)-, -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(DGIu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DGIu)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DGIu)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DGIu)-Arg-Leu-Thr-Glu-,

[0519] -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DGIu)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-?-Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-;-Pro-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu- / -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leii-Glu)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DGIu)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DGIu)-Arg-Arg-Leu-Thr-Glu-;-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-;

[0520] -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu- -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Glu)-Arg-Arg-Leu-Thr-Glu-;-Pro-Ser-Arg-Leu-Glu-cyclofLys-Glu-Leu-DGIuj-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DGlu)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-? -Pro-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-,

[0521] -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-,

[0522] -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-,

[0523] -Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-,

[0524] -Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-,

[0525] -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-,

[0526] -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-,

[0527] -Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-,

[0528] -Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-,

[0529] -Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-,

[0530] -Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-,

[0531] -Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-,

[0532] -Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-,

[0533] -Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-,

[0534] -Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-,

[0535] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-,

[0536] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-,

[0537] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-,

[0538] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-,

[0539] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-,

[0540] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-,

[0541] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-,

[0542] -Pro-SerJ\rg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-,

[0543] -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-,

[0544] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-,

[0545] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Cys)-Glu-,

[0546] -Ser-Arg-Leu-Glu-Glu-cyclofCys-Leu-Arg-CysJ-Arg-Leu-Thr-Glu-,

[0547] -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-,

[0548] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-,

[0549] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Cys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-;-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-;-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-;-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-;-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-;-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-z-Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu- / -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-;

[0550] -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DCys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-Cys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DCys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-Cys)-?

[0551] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-DCys)-,

[0552] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-hCys)-,

[0553] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-,

[0554] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-hCys)-,

[0555] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-,

[0556] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-Cys)-,

[0557] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-DCys)-,

[0558] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-hCys)-,

[0559] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-,

[0560] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DhCys)-,

[0561] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclofCys-Leu-Thr-DhCys)-,

[0562] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-Pen)-,

[0563] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-DPen)-,

[0564] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-Pen)-,

[0565] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-DPen)-,

[0566] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-Cys)-Glu-,

[0567] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-DCys)-Glu-,

[0568] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-DCys)-Glu-,

[0569] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-Cys)-Glu-,

[0570] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DCys)-Glu-,

[0571] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-hCys)-Glu-,

[0572] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-hCys)-Glu-,

[0573] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DhCys)-Glu-,

[0574] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DhCys-Arg-Leu-hCys)-Glu-,

[0575] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DCys)-,

[0576] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-Cys)-,

[0577] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DCys)-,

[0578] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-Cys)-,

[0579] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-DCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-hCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-hCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-;

[0580] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-Cys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-DCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-hCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DhCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-Pen)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-DPen)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-Pen)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-DPen)- / -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-Cys)-Glu-;

[0581] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-DCys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-DCys)-Glu-;

[0582] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-Cys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DCys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-hCys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-hCys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DhCys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DhCys-Arg-Leu-hCys)-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Asp)-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Asp-Arg-Leu-Lys)-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Glu)-Glu-z-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Glu-Arg-Leu-Lys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Asp)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Asp-Arg-Leu-Lys)-Glu-;

[0583] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Glu)-Glu-; -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Glu-Arg-Leu-Lys)-Glu-, -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)- / -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Arg-Cys)-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DGlu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DGIu)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DGIu)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DGIu)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DGIu)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-,

[0584] -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Glu)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DGIu)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DGIu)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Glu)-Arg-Arg-Leu-Thr-Glu-Pro-;-Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DGIu)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DGlu)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-;-Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-;-Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-DLys)-Arg-Leii-Thr-Glii-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-,

[0585] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-,

[0586] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-,

[0587] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-,

[0588] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-,

[0589] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-,

[0590] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-,

[0591] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-,

[0592] -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0593] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)~Pro-,

[0594] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Cys)-Glu-Pro-,

[0595] -Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-Pro-,

[0596] -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0597] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-Pro-,

[0598] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Cys)-Glu-Pro-,

[0599] -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-Pro-,

[0600] -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0601] -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0602] -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0603] -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0604] -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0605] -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0606] -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0607] -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0608] -Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0609] -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0610] -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0611] -Ser-Arg-Leu-Glu-cyclofDhCys-Glu-Leu-CysJ-Arg-Arg-Leu-Thr-Glu-Pro-,

[0612] -Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0613] -Pro-Ser-Arg-Leu-Glu-cydo(DCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-z

[0614] -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0615] -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0616] -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0617] -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-;

[0618] -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-z

[0619] -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0620] -Pro-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0621] -Pro-Ser-Arg-Leu-Glu-cydo(Cys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0622] -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0623] -Pro-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-,

[0624] -Pro-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-;

[0625] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DCys)-Pro-z

[0626] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-Cys)-Pro-,

[0627] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DCys)-Pro-,

[0628] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-Cys)-Pro-,

[0629] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-DCys)-Pro-z

[0630] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-hCys)-Pro-z

[0631] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-Pro-z

[0632] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-hCys)-Pro-,

[0633] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-Pro-,

[0634] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-Cys)-Pro-,

[0635] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-DCys)-Pro-,

[0636] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-hCys)-Pro-z

[0637] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-Pro-;

[0638] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DhCys)-Pro-z

[0639] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-Pro-;

[0640] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-Pen)-Pro-,

[0641] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-DPen)-Pro-,

[0642] -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-Pen)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-DPen)-Pro-;-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-Cys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-DCys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-DCys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-Cys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DCys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-hCys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-hCys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DhCys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DhCys-Arg-Leu-hCys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-Cys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo{hCys-Leu-Thr-Cys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-DCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-hCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-hCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-Cys)-Pro-,

[0643] -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-DCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-hCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DhCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-Pen)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-DPen)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-Pen)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-DPen)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-Cys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-DCys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-DCys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-Cys)-Glii-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DCys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-hCys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-hCys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DhCys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DhCys-Arg-Leu-hCys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Asp)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Asp-Arg-Leu-Lys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Glu)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Glu-Arg-Leu-Lys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Asp)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Asp-Arg-Leu-Lys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Glu)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(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-.

[0644] In some embodiments, the cyclic peptide is attached to a 3-mercaptopropionyl moiety through an a-amine moiety of the leftmost amino acid in the cyclic peptide. In some embodiments, the rightmost amino acid in the cyclic peptide comprises an amide.

[0645] In some embodiments, the cyclized amino acid sequence is one selected from the group consisting of

[0646] -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-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-DCys)-Arg-Arg-Leu-Thr-Glu-, -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)-, -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-.

[0647] In some embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Glu-Glu- Leu-Arg-Lys)-Arg-Leu-Thr-Glu-. In some other embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-. In yet some other embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Lys)- Arg-Arg-Leu-Thr-Glu-. In still some other embodiments, the cyclized amino acid sequence is - Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Glu)-.

[0648] The cyclic peptides may have different cyclic bridging moieties forming the ring structure. Preferably, chemically stable bridging moieties are included in the ring structure such as, for example, an amide group, a lactone group, an ether group, a thioether group, a disulfide group, an alkylene group, an alkenyl group, or a 1,2,3-triazole. The following are examples illustrating the variability of bridging moieties in a peptide:

[0649] In some embodiments, an ALFA-tag binding moiety comprises an antibody or antibody fragment, e.g., a camelid VHH domain. In some embodiments, an ALFA-tag binding moiety comprises a single-domain antibody (sdAb), NbALFA-nanobody.

[0650] In some embodiments, an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence VTXjSALNAMAMG, wherein Xi is I or V, the CDR2 sequence AVSX2RGNAM, wherein X2 is E, H, N, D, or S, and the CDR3 sequence LEDRVDSFHDY.

[0651] In some embodiments, an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence GVTXiSALNAMAMG, wherein Xi is I or V, the CDR2 sequence AVSX2RGNAM, wherein X2 is E, H, N, D, or S, and the CDR3 sequence LEDRVDSFHDY.

[0652] In some embodiments, an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence VTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY.

[0653] In some embodiments, an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence GVTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY.

[0654] In some embodiments, an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the amino acid sequence EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSERGNAMYRESV QGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence, or a fragment of said amino acid sequence or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence. In some embodiments, the amino acid sequence comprises CDR1, CDR2 and CDR3 sequences as described above.

[0655] In some embodiments, the epitope tag / binder system comprises an epitope tag comprising the sequence PDRVRAVSHWSS (Spot-tag) and the binder comprises a single-domain antibody (sdAb, or nanobody) (Spot-nanobody (14.7 kD)) that specifically binds to the Spot-tag.

[0656] In some embodiments, following binding of the moieties on the targeting compound and on the docking compound interacting which each other, a covalent connection is formed. In these embodiments, the system used herein may comprise a Tag / Catcher system forming a covalent bond, e.g., SpyTag / SpyCatcher forming an isopeptide bond.

[0657] The SpyTag / SpyCatcher system is a technology for irreversible conjugation of recombinant proteins. The peptide SpyTag spontaneously reacts with the protein SpyCatcher to form an intermolecular isopeptide bond between the pair. Using the Tag / Catcher pair, bioconjugation can be achieved between two recombinant proteins. The present disclosure provides in one aspect, a complex wherein a particle comprising a targeting compound is bound to a docking compound. Thus, the targeting compound and the docking compound comprise moieties interacting which each other.

[0658] Different embodiments of the targeting compound and the docking compound which are complexed are described herein.

[0659] In some embodiments, the targeting compound comprises an ALFA-tag. In these embodiments, the moiety binding to a targeting compound of the docking compound may be a NbALFA-nanobody (NbALFA). In some embodiments, the docking compound may have a structure selected from the group consisting of NbALFA x anti-primary target DARPin, NbALFA x anti-primary target VHH and NbALFA x anti-primary target scFv.

[0660] Embodiments of targeting compound

[0661] In some embodiments, the targeting compound described herein is a lipid-PEG-peptide conjugate compound having the structure (I): wherein the Lipid is a phospholipid attached to the carbonyl through the amino group of the ethanolamine moiety thereof, PEG has a molecular weight of from about 130 to about 50,000, a-amino group of the left most amino acid group of the Peptide is attached to the carbonyl group of the 3-mercaptopropionyl moiety and the Peptide comprises from about 11 to about 15 amino acids, and Z is a bond or -CH2-. In some embodiments, the lipid-PEG-peptide conjugate has the following structure (II)

[0662]

[0663] A particular embodiment of the lipid-PEG-peptide conjugate compound having the 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 a cyclic peptide selected from the group consisting of -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Ser-Arg- Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu- Lys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Glu)-, - Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Asp- Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr- Asp)-, -Pro-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu- Glu-cyclo(DGIu-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-, -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-.

[0664] The term "PEG" as used in the above formula I means 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 the PEG is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy or aryl groups. In one embodiment, the PEG has a molecular weight of from about 130 to about 50,000, in another embodiment about 150 to about 30,000, in another embodiment about 150 to about 20,000, in another embodiment about 150 to about 15,000, in another embodiment about 150 to about 10,000, in another embodiment about 150 to about 6000, in another embodiment about 150 to about 5000, in another embodiment about 150 to about 4000, in another embodiment about 150 to about 3000, in another embodiment about 300 to about 3000, in another embodiment about 1000 to about 3000, and in still another embodiment about 1500 to about 2500.

[0665] In certain embodiments the PEG (conjugated to the lipid) is a "PEG2k", also termed "PEG 2000", which has an average molecular weight of about 2000 Daltons. Another name for a lipid comprising polyethylene glycol PEG, such as, for example PEG2K, is a "pegylated lipid" and, if a phosphorous-containing linkage is present, the lipid is generally referred to herein as a "phospholipid" or a "pegylated phospholipid."

[0666] In some embodiments, the lipid portion to which the PEG is bonded in the functionalized stealth lipid disclosed in the above formula I comprises a neutral phospholipid. Examples of neutral phospholipids include, but are not limited to: dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoyl-sn-glycero-3-phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn- glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1- myristoyl-2-palmitoyl phosphatidylcholine (MPPC), l-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), l-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2- diarachidoyl-sn-glycero-3-phosphocholine (DBPC), l-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), l,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphophatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In certain embodiments, a preferred lipid is distearoyl-phosphatidylethanolamine (DSPE).

[0667] The term "PEPTIDE" or "Peptide" are used interchangeably and as used in the above formula I or II refers to a series of amino acids connected one to another by peptide bonds between the amino and carboxy groups of adjacent residues. "PEPTIDE-NH2" represents that the C- terminus carboxyl group of the peptide is an amide. In some embodiments, the term "PEPTIDE" or "Peptide" refers to an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein.

[0668] Particles comprising an RNA payload

[0669] The particles described herein comprise one or more particle forming agents, an RNA payload to be delivered to a target immune cell and a targeting compound for binding to the target immune cells or the docking compound.

[0670] RNA payload

[0671] In some embodiments, an RNA payload is delivered to target immune cells to transfect the target immune cells and enable the target immune cells to express a cytokine or a functional variant thereof encoded by the RNA.

[0672] Examples of cytokines include interferons, such as interferon-alpha (IFN-a) or interferongamma ( I FN-y), interleukins, such as IL-2, IL-7, IL-12, IL-15 and IL-21, colony stimulating factors, such as M-CSF and GM-CSF, and tumor necrosis factor.

[0673] In some embodiments, the cytokine is involved in and preferably induces or enhances development, priming, expansion, differentiation and / or survival of T cells. In some embodiments, the cytokine is an interleukin. In some embodiments, the cytokine is an interleukin selected from the group consisting of IL-2, IL-7, IL-12, IL-15, and IL-21.

[0674] The term "cytokines" relates to proteins which have a molecular weight of about 5 to 60 kDa and which participate in cell signaling (e.g., paracrine, endocrine, and / or autocrine signaling). In particular, when released, cytokines exert an effect on the behavior of cells around the place of their release. Examples of cytokines include lymphokines, interleukins, chemokines, interferons, and tumor necrosis factors (TNFs). According to the present disclosure, cytokines do not include hormones or growth factors. Cytokines differ from hormones in that (i) they usually act at much more variable concentrations than hormones and (ii) generally are made by a broad range of cells (nearly all nucleated cells can produce cytokines). Particular examples of cytokines include erythropoietin (EPO), colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF), bone morphogenetic protein (BMP), interferon alfa (IFN-a), interferon beta (IFN-p), interferon gamma (INF-y), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 10 (IL-10), interleukin 11 (IL-11), interleukin 12 (IL-12), interleukin 15 (IL-15), and interleukin 21 (IL-21), as well as variants and derivatives thereof.

[0675] According to the disclosure, a cytokine may be a naturally occurring cytokine or a functional fragment or variant thereof. A cytokine may be human cytokine and may be derived from any vertebrate, especially any mammal. One particularly preferred cytokine is IL-2 or a functional fragment or variant thereof.

[0676] In some embodiments, a suitable cytokine for use herein is a cytokine involved in T cell proliferation and / or maintenance. Examples of suitable cytokines include IL-2 or IL-7, fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants, such as extended-PK cytokines.

[0677] In some embodiments, a suitable cytokine for use herein is a cytokine involved in inducing an immune response, in particular in T-cell priming or activation of resident immune cells.

[0678] Examples of cytokines involved in T cell priming include IL-12, IL-15, IFN-a, or IFN-P, fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants, such as extended-PK cytokines.

[0679] Interferons (IFNs) are a group of signaling proteins made and released by host cells in response to the presence of several pathogens, such as viruses, bacteria, parasites, and also tumor cells. In a typical scenario, a virus-infected cell will release interferons causing nearby cells to heighten their anti-viral defenses. Interferons are usually characterized by antiviral, antiproliferative and immunomodulatory activities. Interferons are proteins that alter and regulate the transcription of genes within a cell by binding to interferon receptors on the regulated cell's surface, thereby preventing viral replication within the cells.

[0680] Based on the type of receptor through which they signal, interferons are typically divided among three classes: type I interferon, type II interferon, and type III interferon.

[0681] All type I interferons bind to a specific cell surface receptor complex known as the IFN-a / P receptor (IFNAR) that consists of IFNAR1 and IFNAR2 chains.

[0682] The type I interferons present in humans are IFNa, I FNP, I FNe, IFNK and IFNu). In general, type I interferons are produced when the body recognizes a virus that has invaded it. They are produced by fibroblasts and monocytes. Once released, type I interferons bind to specific receptors on target cells, which leads to expression of proteins that will prevent the virus from producing and replicating its RNA and DNA.

[0683] The IFN-a proteins are produced mainly by plasmacytoid dendritic cells (pDCs). They are mainly involved in innate immunity against viral infection. The genes responsible for their synthesis come in 13 subtypes that are called IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21. These genes are found together in a cluster on chromosome 9.

[0684] The IFN-P proteins are produced in large quantities by fibroblasts. They have antiviral activity that is involved mainly in innate immune response. Two types of IFN-P have been described, IFN-pi and IFN-P3. The natural and recombinant forms of IFN-pl have antiviral, antibacterial, and anticancer properties.

[0685] Type II interferon (IFN-y in humans) is also known as immune interferon and is activated by I L12. Furthermore, type II interferons are released by cytotoxic T cells and T helper cells.

[0686] Type III interferons signal through a receptor complex consisting of IL10R2 (also called CRF2- 4) and IFNLR1 (also called CRF2-12). Although discovered more recently than type I and type II IFNs, recent information demonstrates the importance of type III IFNs in some types of virus or fungal infections.

[0687] In general, type I and II interferons are responsible for regulating and activating the immune response.

[0688] According to the disclosure, a type I interferon is preferably IFN-a or IFN-P, more preferably IFN-a.

[0689] According to the disclosure, an interferon may be a naturally occurring interferon or a functional fragment or variant thereof. An interferon may be human interferon and may be derived from any vertebrate, especially any mammal.

[0690] Interleukins (ILs) are a group of cytokines (secreted proteins and signal molecules) that can be divided into four major groups based on distinguishing structural features. However, their amino acid sequence similarity is rather weak (typically 15-25% identity). The human genome encodes more than 50 interleukins and related proteins. According to the disclosure, an interleukin may be a naturally occurring interleukin or a functional fragment or variant thereof. An interleukin may be human interleukin and may be derived from any vertebrate, especially any mammal.

[0691] Cytokine polypeptides described herein can be prepared as fusion or chimeric polypeptides that include a portion comprising a cytokine or a functional variant thereof and a heterologous polypeptide (i.e., a polypeptide that is not a cytokine or a functional variant thereof). A cytokine or functional variant thereof may be fused to an extended-PK group, which increases circulation half-life. Non-limiting examples of extended-PK groups are described infra. It should be understood that other PK groups that increase the circulation half-life of cytokines, or variants thereof, are also applicable to the present disclosure. In certain embodiments, the extended-PK group is a serum albumin domain (e.g., mouse serum albumin, human serum albumin).

[0692] As used herein, the term "PK" is an acronym for "pharmacokinetic" and encompasses properties of a compound including, by way of example, absorption, distribution, metabolism, and elimination by a subject. As used herein, an "extended-PK group" refers to a protein, peptide, or moiety that increases the circulation half-life of a biologically active molecule when fused to or administered together with the biologically active molecule. Examples of an extended-PK group include serum albumin (e.g., HSA), Immunoglobulin Fc or Fc fragments and variants thereof, transferrin and variants thereof, and human serum albumin (HSA) binders (as disclosed in U.S. Publication Nos. 2005 / 0287153 and 2007 / 0003549). Other exemplary extended-PK groups are disclosed in Kontermann, Expert Opin Biol Ther, 2016 Jul; 16(7):903- 15 which is herein incorporated by reference in its entirety. As used herein, an "extended-PK" cytokine refers to a cytokine moiety (including functional variants thereof) in combination with an extended-PK group. In some embodiments, the extended-PK cytokine is a fusion protein in which a cytokine moiety is linked or fused to an extended-PK group.

[0693] In certain embodiments, the serum half-life of an extended-PK cytokine is increased relative to the cytokine alone (i.e., the cytokine not fused to an extended-PK group). In certain embodiments, the serum half-life of the extended-PK cytokine is at least 20%, at least 40%, at least 60%, at least 80%, at least 100%, at least 120%, at least 150%, at least 180%, at least 200%, at least 400%, at least 600%, at least 800%, or at least 1000% longer relative to the serum half-life of the cytokine alone. In certain embodiments, the serum half-life of the extended-PK cytokine is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5- fold, 6-fold, 7-fold, 8-fold, 10-fold, 12-fold, 13-fold, 15-fold, 17-fold, 20-fold, 22-fold, 25-fold, 27-fold, 30-fold, 35-fold, 40-fold, or 50-fold greater than the serum half-life of the cytokine alone. In certain embodiments, the serum half-life of the extended-PK cytokine is at least 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200 hours.

[0694] As used herein, "half-life" refers to the time taken for the serum or plasma concentration of a compound such as a peptide or polypeptide to reduce by 50%, in vivo, for example due to degradation and / or clearance or sequestration by natural mechanisms. An extended-PK cytokine suitable for use herein is stabilized in vivo and its half-life increased by, e.g., fusion to serum albumin (e.g., HSA or MSA), which resist degradation and / or clearance or sequestration. The half-life can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art, and may for example generally involve the steps of suitably administering a suitable dose of the amino acid sequence or compound to a subject; collecting blood samples or other samples from said subject at regular intervals; determiningthe level or concentration of the amino acid sequence or compound in said blood sample; and calculating, from (a plot of) the data thus obtained, the time until the level or concentration of the amino acid sequence or compound has been reduced by 50% compared to the initial level upon dosing. Further details are provided in, e.g., standard handbooks, such as Kenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al., Pharmacokinetic Analysis: A Practical Approach (1996). Reference is also made to Gibaldi, M. et al., Pharmacokinetics, 2nd Rev. Edition, Marcel Dekker (1982).

[0695] In certain embodiments, the extended-PK group includes serum albumin, or fragments thereof or variants of the serum albumin or fragments thereof (all of which for the purpose of the present disclosure are comprised by the term "albumin"). Polypeptides described herein may be fused to albumin (or a fragment or variant thereof) to form albumin fusion proteins. Such albumin fusion proteins are described in U.S. Publication No. 20070048282. As used herein, "albumin fusion protein" refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) to at least one molecule of a protein such as a therapeutic protein, in particular a cytokine. The albumin fusion protein may be generated by translation of a nucleic acid in which a polynucleotide encoding a therapeutic protein is joined in-frame with a polynucleotide encoding an albumin. The therapeutic protein and albumin, once part of the albumin fusion protein, may each be referred to as a "portion", "region” or "moiety” of the albumin fusion protein (e.g., a "therapeutic protein portion" or an "albumin protein portion"). In a highly preferred embodiment, an albumin fusion protein comprises at least one molecule of a therapeutic protein (including, but not limited to a mature form of the therapeutic protein) and at least one molecule of albumin (including but not limited to a mature form of albumin). In some embodiments, an albumin fusion protein is processed by a host cell such as a cell of the target organ for administered RNA, and secreted into the circulation. Processing of the nascent albumin fusion protein that occurs in the secretory pathways of the host cell used for expression of the RNA may include, but is not limited to signal peptide cleavage; formation of disulfide bonds; proper folding; addition and processing of carbohydrates (such as for example, N- and O-linked glycosylation); specific proteolytic cleavages; and / or assembly into multimeric proteins. An albumin fusion protein is preferably encoded by RNA in a non-processed form which in particular has a signal peptide at its N-terminus and following secretion by a cell is preferably present in the processed form wherein in particular the signal peptide has been cleaved off. In a most preferred embodiment, the "processed form of an albumin fusion protein" refers to an albumin fusion protein product which has undergone N-terminal signal peptide cleavage, herein also referred to as a "mature albumin fusion protein".

[0696] In preferred embodiments, albumin fusion proteins comprising a therapeutic protein have a higher plasma stability compared to the plasma stability of the same therapeutic protein when not fused to albumin. Plasma stability typically refers to the time period between when the therapeutic protein is administered in vivo and carried into the bloodstream and when the therapeutic protein is degraded and cleared from the bloodstream, into an organ, such as the kidney or liver, that ultimately clears the therapeutic protein from the body. Plasma stability is calculated in terms of the half-life of the therapeutic protein in the bloodstream. The half- life of the therapeutic protein in the bloodstream can be readily determined by common assays known in the art.

[0697] As used herein, "albumin" refers collectively to albumin protein or amino acid sequence, or an albumin fragment or variant, having one or more functional activities (e.g., biological activities) of albumin. In particular, "albumin" refers to human albumin or fragments or variants thereof especially the mature form of human albumin, or albumin from other vertebrates or fragments thereof, or variants of these molecules. The albumin may be derived from any vertebrate, especially any mammal, for example human, cow, sheep, or pig. Nonmammalian albumins include, but are not limited to, hen and salmon. The albumin portion of the albumin fusion protein may be from a different animal than the therapeutic protein portion.

[0698] In certain embodiments, the albumin is human serum albumin (HSA), or fragments or variants thereof, such as those disclosed in US 5,876,969, WO 2011 / 124718, WO 2013 / 075066, and WO 2011 / 0514789.

[0699] The terms, human serum albumin (HSA) and human albumin (HA) are used interchangeably herein. The terms, "albumin and "serum albumin" are broader, and encompass human serum albumin (and fragments and variants thereof) as well as albumin from other species (and fragments and variants thereof).

[0700] As used herein, a fragment of albumin sufficient to prolong the therapeutic activity or plasma stability of the therapeutic protein refers to a fragment of albumin sufficient in length or structure to stabilize or prolong the therapeutic activity or plasma stability of the protein so that the plasma stability of the therapeutic protein portion of the albumin fusion protein is prolonged or extended compared to the plasma stability in the non-fusion state.

[0701] The albumin portion of the albumin fusion proteins may comprise the full length of the albumin sequence, or may include one or more fragments thereof that are capable of stabilizing or prolonging the therapeutic activity or plasma stability. Such fragments may be of 10 or more amino acids in length or may include about 15, 20, 25, 30, 50, or more contiguous amino acids from the albumin sequence or may include part or all of specific domains of albumin. For instance, one or more fragments of HSA spanning the first two immunoglobulin- like domains may be used. In a preferred embodiment, the HSA fragment is the mature form of HSA.

[0702] Generally speaking, an albumin fragment or variant will be at least 100 amino acids long, preferably at least 150 amino acids long.

[0703] According to the disclosure, albumin may be naturally occurring albumin or a fragment or variant thereof. Albumin may be human albumin and may be derived from any vertebrate, especially any mammal.

[0704] Preferably, the albumin fusion protein comprises albumin as the N-terminal portion, and a therapeutic protein as the C-terminal portion. Alternatively, an albumin fusion protein comprising albumin as the C-terminal portion, and a therapeutic protein as the N-terminal portion may also be used. In other embodiments, the albumin fusion protein has a therapeutic protein fused to both the N-terminus and the C-terminus of albumin. In a preferred embodiment, the therapeutic proteins fused at the N- and C-termini are the same therapeutic proteins. In another preferred embodiment, the therapeutic proteins fused at the N- and C- termini are different therapeutic proteins. In some embodiments, the different therapeutic proteins are both cytokines.

[0705] In some embodiments, the therapeutic protein(s) is (are) joined to the albumin through (a) peptide linker(s). A peptide linker between the fused portions may provide greater physical separation between the moieties and thus maximize the accessibility of the therapeutic protein portion, for instance, for binding to its cognate receptor. The peptide linker may consist of amino acids such that it is flexible or more rigid. The linker sequence may be cleavable by a protease or chemically.

[0706] As used herein, the term "Fc region" refers to the portion of a native immunoglobulin formed by the respective Fc domains (or Fc moieties) of its two heavy chains. As used herein, the term "Fc domain" refers to a portion or fragment of a single immunoglobulin (Ig) heavy chain wherein the Fc domain does not comprise an Fv domain. In certain embodiments, an Fc domain begins in the hinge region just upstream of the papain cleavage site and ends at the C-terminus of the antibody. Accordingly, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments, an Fc domain comprises at least one of: a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragment thereof. In certain embodiments, an Fc domain comprises a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In certain embodiments, an Fc domain comprises a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof). In certain embodiments, an Fc domain comprises a CH2 domain (or portion thereof) fused to a CH3 domain (or portion thereof). In certain embodiments, an Fc domain consists of a CH3 domain or portion thereof. In certain embodiments, an Fc domain consists of a hinge domain (or portion thereof) and a CH3 domain (or portion thereof). In certain embodiments, an Fc domain consists of a CH2 domain (or portion thereof) and a CH3 domain. In certain embodiments, an Fc domain consists of a hinge domain (or portion thereof) and a CH2 domain (or portion thereof). In certain embodiments, an Fc domain lacks at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). An Fc domain herein generally refers to a polypeptide comprising all or part of the Fc domain of an immunoglobulin heavy-chain. This includes, but is not limited to, polypeptides comprising the entire CHI, hinge, CH2, and / or CH3 domains as well as fragments of such peptides comprising only, e.g., the hinge, CH2, and CH3 domain. The Fc domain may be derived from an immunoglobulin of any species and / or any subtype, including, but not limited to, a human IgGl, lgG2, lgG3, lgG4, IgD, IgA, IgE, or IgM antibody. The Fc domain encompasses native Fc and Fc variant molecules. As set forth herein, it will be understood by one of ordinary skill in the art that any Fc domain may be modified such that it varies in amino acid sequence from the native Fc domain of a naturally occurring immunoglobulin molecule. In certain embodiments, the Fc domain has reduced effector function (e.g., FcyR binding).

[0707] The Fc domains of a polypeptide described herein may be derived from different immunoglobulin molecules. For example, an Fc domain of a polypeptide may comprise a CH2 and / or CH3 domain derived from an IgGl molecule and a hinge region derived from an lgG3 molecule. In another example, an Fc domain can comprise a chimeric hinge region derived, in part, from an IgGl molecule and, in part, from an lgG3 molecule. In another example, an Fc domain can comprise a chimeric hinge derived, in part, from an IgGl molecule and, in part, from an lgG4 molecule.

[0708] In certain embodiments, an extended-PK group includes an Fc domain or fragments thereof or variants of the Fc domain or fragments thereof (all of which for the purpose of the present disclosure are comprised by the term "Fc domain"). The Fc domain does not contain a variable region that binds to antigen. Fc domains suitable for use in the present disclosure may be obtained from a number of different sources. In certain embodiments, an Fc domain is derived from a human immunoglobulin. In certain embodiments, the Fc domain is from a human IgGl constant region. It is understood, however, that the Fc domain may be derived from an immunoglobulin of another mammalian species, including for example, a rodent (e.g. a mouse, rat, rabbit, guinea pig) or non-human primate (e.g. chimpanzee, macaque) species.

[0709] Moreover, the Fc domain (or a fragment or variant thereof) may be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgGl, lgG2, lgG3, and lgG4.

[0710] A variety of Fc domain gene sequences (e.g., mouse and human constant region gene sequences) are available in the form of publicly accessible deposits. Constant region domains comprising an Fc domain sequence can be selected lacking a particular effector function and / or with a particular modification to reduce immunogenicity. Many sequences of antibodies and antibody-encoding genes have been published and suitable Fc domain sequences (e.g. hinge, CH2, and / or CH3 sequences, or fragments or variants thereof) can be derived from these sequences using art recognized techniques.

[0711] In certain embodiments, the extended-PK group is a serum albumin binding protein such as those described in US2005 / 0287153, US2007 / 0003549, US2007 / 0178082, US2007 / 0269422, US2010 / 0113339, W02009 / 083804, and W02009 / 133208, which are herein incorporated by reference in their entirety. In certain embodiments, the extended-PK group is transferrin, as disclosed in US 7,176,278 and US 8,158,579, which are herein incorporated by reference in their entirety. In certain embodiments, the extended-PK group is a serum immunoglobulin binding protein such as those disclosed in US2007 / 0178082, US2014 / 0220017, and US2017 / 0145062, which are herein incorporated by reference in their entirety. In certain embodiments, the extended-PK group is a fibronectin (Fn)-based scaffold domain protein that binds to serum albumin, such as those disclosed in US2012 / 0094909, which is herein incorporated by reference in its entirety. Methods of making fibronectin-based scaffold domain proteins are also disclosed in US2012 / 0094909. A non-limiting example of a Fn3-based extended-PK group is Fn3(HSA), i.e., a Fn3 protein that binds to human serum albumin. In certain aspects, the extended-PK cytokine, suitable for use according to the disclosure, can employ one or more peptide linkers. As used herein, the term "peptide linker" refers to a peptide or polypeptide sequence which connects two or more domains (e.g., the extended- PK moiety and a cytokine moiety) in a linear amino acid sequence of a polypeptide chain. For example, peptide linkers may be used to connect a cytokine moiety to a HSA domain.

[0712] Linkers suitable for fusing the extended-PK group to, e.g., a cytokine are well known in the art. Exemplary linkers include glycine-serine-polypeptide linkers, glycine-proline-polypeptide linkers, and proline-alanine polypeptide linkers. In certain embodiments, the linker is a glycine-serine-polypeptide linker, i.e., a peptide that consists of glycine and serine residues.

[0713] A particularly preferred cytokine for use herein is IL-2 or a functional variant thereof. Human IL-2 is a key cytokine in T cell immunity. It supports the differentiation, proliferation, survival and effector functions of T cells (Gillis S, Smith KA, Nature 1977; 268(5616): 154-56, Blattman JN et al., Nat Med 2003; 9(5): 540-47, Bamford RN et al., Proc Natl Acad Sci USA. 1994; 91(11): 4940-44, Kamimura D, Bevan MJ, J Exp Med 2007; 204(8): 1803-12). Recombinant rlL-2, aldesleukin, was the first approved cancer immunotherapy and has been used for decades in the treatment of late stage malignant melanoma and renal cell cancer (Kammula US et al., Cancer 1998; 83(4): 797-805). Most patients with complete responses after rlL-2 treatment remain regression free for more than 25 years after initial treatment, but overall response rates are low (Klapper JA et aL, Cancer 2008; 113(2): 293-301, Rosenberg SA et al., Ann Surg 1998; 228(3): 307-19). A particular challenge of rl L2 for cancer treatment is its very short halflife and its side effects.

[0714] According to the disclosure, human IL-2 (hlL-2) (optionally as a portion of extended-PK hlL-2) may be naturally occurring hlL-2 or a fragment or variant thereof. In one embodiment, hlL-2 comprises the following amino acid sequence, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the following amino acid sequence, or a functional fragment of the following amino acid sequence, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the following amino acid sequence. In one embodiment, hlL-2 or a h IL-2 fragment or variant binds to the IL-2 receptor. APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNL

[0715] AQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0716] In one embodiment, hlL-2 comprises the following amino acid sequence, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the following amino acid sequence, or a functional fragment of the following amino acid sequence, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the following amino acid sequence. In one embodiment, hlL-2 or a hlL-2 fragment or variant binds to the IL-2 receptor.

[0717] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKAT ELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFC QSIISTLT

[0718] Particles

[0719] An RNA payload may be administered with one or more delivery vehicles that protect the payload from degradation, maximize delivery to on-target cells and minimize exposure to off- target cells. Such delivery vehicles may complex or encapsulate the payload and include a range of materials, including polymers, lipids and mixtures thereof. In some embodiments, such delivery vehicles may form particles with the payload.

[0720] In the context of the present disclosure, the term "particle" relates to a structured entity formed by molecules or molecule complexes, in particular particle forming compounds. In some embodiments, a particle is a nucleic acid containing particle such as a particle comprising DNA, RNA or a mixture thereof. In some embodiments, the particle contains an envelope (e.g., one or more layers or lamellas) made of one or more types of amphiphilic substances (e.g., amphiphilic lipids). In this context, the expression "amphiphilic substance" means that the substance possesses both hydrophilic and lipophilic properties. The envelope may also comprise additional substances (e.g., additional lipids) which do not have to be amphiphilic. Thus, the particle may be a monolamellar or multilamellar structure, wherein the substances constituting the one or more layers or lamellas comprise one or more types of amphiphilic substances (in particular selected from the group consisting of amphiphilic lipids) optionally in combination with additional substances (e.g., additional lipids) which do not have to be amphiphilic. In some embodiments, the term "particle" relates to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure. According to the present disclosure, the term "particle" includes nanoparticles.

[0721] The term "nanoparticle" relates to a nano-sized particle comprising at least one particle forming agent, e.g., at least one cationic or cationically ionizable lipid, wherein all three external dimensions of the particle are in the nanoscale, i.e., at least about 1 nm and below about 1000 nm. Preferably, the size of a particle is its diameter.

[0722] In some embodiments, the particles described herein have a size (such as a diameter) in the range of about 10 to about 2000 nm, such as at least about 15 nm (e.g., at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, or at least about 100 nm) and / or at most about 1900 nm (e.g., at most about 1800 nm, at most about 1700 nm, at most about 1600 nm, at most about 1500 nm, at most about 1400 nm, at most about 1300 nm, at most about 1200 nm, at most 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), such as in the range of about 20 to about 1500 nm, such as about 30 to about 1200 nm, about 40 to about 1100 nm, about 50 to about 1000 nm, about 60 to about 900 nm, about 70 to about 800 nm, about 80 to about 700 nm, about 90 to about 600 nm, or about 50 to about 500 nm or about 100 to about 500 nm, such as in the range of 10 to 1000 nm, 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, 50 to 250 nm, 60 to 200 nm, 70 to 150 nm, or 80 to 150 nm. In some embodiments, the particles described herein have a size (such as a diameter) in the range of from about 40 nm to about 200 nm, such as from about 50 nm to about 180 nm, from about 60 nm to about 160 nm, from about 80 nm to about 150 nm or from about 80 nm to about 120 nm.

[0723] In some embodiments, the particles described herein have an average diameter that in some embodiments ranges from about 50 nm to about 1000 nm, from about 50 nm to about 800 nm, from about 50 nm to about 700 nm, from about 50 nm to about 600 nm, from about 50 nm to about 500 nm, from about 50 nm to about 450 nm, from about 50 nm to about 400 nm, from about 50 nm to about 350 nm, from about 50 nm to about 300 nm, from about 50 nm to about 250 nm, from about 50 nm to about 200 nm, from about 100 nm to about 1000 nm, from about 100 nm to about 800 nm, from about 100 nm to about 700 nm, from about 100 nm to about 600 nm, from about 100 nm to about 500 nm, from about 100 nm to about 450 nm, from about 100 nm to about 400 nm, from about 100 nm to about 350 nm, from about 100 nm to about 300 nm, from about 100 nm to about 250 nm, from about 100 nm to about 200 nm, from about 150 nm to about 1000 nm, from about 150 nm to about 800 nm, from about 150 nm to about 700 nm, from about 150 nm to about 600 nm, from about 150 nm to about 500 nm, from about 150 nm to about 450 nm, from about 150 nm to about 400 nm, from about 150 nm to about 350 nm, from about 150 nm to about 300 nm, from about 150 nm to about 250 nm, from about 150 nm to about 200 nm, from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 200 nm to about 700 nm, from about 200 nm to about 600 nm, from about 200 nm to about 500 nm, from about 200 nm to about 450 nm, from about 200 nm to about 400 nm, from about 200 nm to about 350 nm, from about 200 nm to about 300 nm, from about 200 nm to about 250 nm, or from about 80 to about 150 nm. In some embodiments, the particles described herein have an average diameter that in some embodiments ranges from about 40 nm to about 200 nm, such as from about 50 nm to about 180 nm, from about 60 nm to about 160 nm, from about 80 nm to about 150 nm or from about 80 nm to about 120 nm.

[0724] Particles described herein may exhibit a polydispersity index (PDI) less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1, or less than about 0.05. By way of example, the particles can exhibit a polydispersity index in a range of about 0.01 to about 0.4 or about 0.1 to about 0.3.

[0725] A "nucleic acid particle" can be used to deliver nucleic acid to a target site of interest (e.g., cell, tissue, organ, and the like). A nucleic acid particle may be formed from at least one cationic or cationically ionizable compound such as a polymer or lipid complexing the nucleic acid. Without intending to be bound by any theory, it is believed that the cationic or cationica I ly ionizable compound combines together with the nucleic acid to form aggregates, and this aggregation results in colloidally stable particles.

[0726] In some embodiments, nucleic acid may be noncovalently associated with a particle. In some embodiments, the nucleic acid may be adhered to the outer surface of the particle (surface nucleic acid) and / or may be contained in the particle (encapsulated nucleic acid).

[0727] The N / P ratio gives the ratio of the nitrogen groups in the lipid to the number of phosphate groups in the nucleic acid. It is correlated to the charge ratio, as the nitrogen atoms (depending on the pH) are usually positively charged and the phosphate groups are negatively charged. The N / P ratio, where a charge equilibrium exists, depends on the pH. Lipid formulations are frequently formed at N / P ratios larger than four up to twelve, because positively charged nanoparticles are considered favorable for transfection. In that case, nucleic acid is considered to be completely bound to nanoparticles.

[0728] Particles described herein can be prepared using a wide range of methods. For example, methods for preparing nucleic acid particles may involve obtaining a colloid from at least one cationic or cationically ionizable lipid and mixing the colloid with nucleic acid to obtain nucleic acid particles.

[0729] The term "colloid" as used herein relates to a type of homogeneous mixture in which dispersed particles do not settle out. The insoluble particles in the mixture are microscopic, with particle sizes between 1 and 1000 nanometers. The mixture may be termed a colloid or a colloidal suspension. Sometimes the term "colloid" only refers to the particles in the mixture and not the entire suspension.

[0730] For the preparation of colloids comprising at least one cationic or cationically ionizable lipid methods are applicable herein that are conventionally used for preparing liposomal vesicles and are appropriately adapted. The most commonly used methods for preparing liposomal vesicles share the following fundamental stages: (i) lipids dissolution in organic solvents, (ii) drying of the resultant solution, and (iii) hydration of dried lipid (using various aqueous media). In the film hydration method, lipids are firstly dissolved in a suitable organic solvent, and dried down to yield a thin film at the bottom of the flask. The obtained lipid film is hydrated using an appropriate aqueous medium to produce a liposomal dispersion. Furthermore, an additional downsizing step may be included. Reverse phase evaporation is an alternative method to the film hydration for preparing liposomal vesicles that involves formation of a water-in-oil emulsion between an aqueous phase and an organic phase containing lipids. A brief sonication of this mixture is required for system homogenization. The removal of the organic phase under reduced pressure yields a milky gel that turns subsequently into a liposomal suspension.

[0731] The term "ethanol injection technique" refers to a process, in which an ethanol solution comprising lipids is rapidly injected into an aqueous solution through a needle. This action disperses the lipids throughout the solution and promotes lipid structure formation, for example lipid vesicle formation such as liposome formation. Generally, lipoplex particles are obtainable by adding nucleic acid to a colloidal liposome dispersion. Using the ethanol injection technique, such colloidal liposome dispersion is, in some embodiments, formed as follows: an ethanol solution comprising lipids, such as cationic or cationically ionizable lipids and additional lipids, is injected into an aqueous solution under stirring.

[0732] The term "particle forming components" or "particle forming agents" relates to any components which form particles, e.g., by associating with a payload. Delivery vehicles such as particle forming agents useful herein include polymers, polymer derivatives, lipids, e.g., as described herein, and mixtures thereof. Such components include any component which can be part of nucleic acid particles, e.g., cationic or cationically ionizable lipids.

[0733] Polymers

[0734] Given their high degree of chemical flexibility, polymers are commonly used materials for nanoparticle-based delivery. Typically, cationic polymers are used to electrostatically condense negatively charged nucleic acid into nanoparticles. These positively charged groups often consist of amines that change their state of protonation in the pH range between 5.5 and 7.5, thought to lead to an ion imbalance that results in endosomal rupture. Polymers such as poly-L-lysine, polyamidoamine, protamine and polyethyleneimine, as well as naturally occurring polymers such as chitosan have all been applied to nucleic acid delivery and are suitable as cationic polymers herein. In addition, some investigators have synthesized polymers specifically for nucleic acid delivery. Polyfp-amino esters), in particular, have gained widespread use in nucleic acid delivery owing to their ease of synthesis and biodegradability. Such synthetic polymers are also suitable as cationic polymers herein.

[0735] A "polymer," as used herein, is given its ordinary meaning, i.e., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds. The repeat units can all be identical, or in some cases, there can be more than one type of repeat unit present within the polymer. In some cases, the polymer is biologically derived, i.e., a biopolymer such as a protein. In some cases, additional moieties can also be present in the polymer, for example targeting moieties.

[0736] If more than one type of repeat unit is present within the polymer, then the polymer is said to be a "copolymer." It is to be understood that the polymer being employed herein can be a copolymer. The repeat units forming the copolymer can be arranged in any fashion. For example, the repeat units can be arranged in a random order, in an alternating order, or as a "block" copolymer, i.e., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc. Block copolymers can have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks.

[0737] In certain embodiments, the polymer is biocompatible. Biocompatible polymers are polymers that typically do not result in significant cell death at moderate concentrations. In certain embodiments, the biocompatible polymer is biodegradable, i.e., the polymer is able to degrade, chemically and / or biologically, within a physiological environment, such as within the body.

[0738] In certain embodiments, polymer may be protamine or polyalkyleneimine.

[0739] The term "protamine" refers to any of various strongly basic proteins of relatively low molecular weight that are rich in arginine and are found associated especially with DNA in place of somatic histones in the sperm cells of various animals (as fish). In particular, the term "protamine" refers to proteins found in fish sperm that are strongly basic, are soluble in water, are not coagulated by heat, and yield chiefly arginine upon hydrolysis. In purified form, they are used in a long-acting formulation of insulin and to neutralize the anticoagulant effects of heparin. According to the disclosure, the term "protamine" as used herein is meant to comprise any protamine amino acid sequence obtained or derived from natural or biological sources including fragments thereof and multimeric forms of said amino acid sequence or fragment thereof as well as (synthesized) polypeptides which are artificial and specifically designed for specific purposes and cannot be isolated from native or biological sources.

[0740] In some embodiments, the polyalkyleneimine comprises polyethylenimine and / or polypropylenimine, preferably polyethyleneimine. A preferred polyalkyleneimine is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75-102to 107Da, preferably 1000 to 105Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, even more preferably 20000 to 25000 Da.

[0741] Preferred according to the disclosure is linear polyalkyleneimine such as linear polyethyleneimine (PEI).

[0742] Cationic polymers (including polycationic polymers) contemplated for use herein include any cationic polymers which are able to electrostatically bind nucleic acid. In some embodiments, cationic polymers contemplated for use herein include any cationic polymers with which nucleic acid can be associated, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.

[0743] In some embodiments, the particle comprises a cationic polymer, e.g., a polycationic polymer, as particle forming component. In some embodiments, a targeting compound is incorporated into the particle comprising a cationic polymer through a negative charge in the moiety incorporating the targeting compound into the particle interacting with a positive charge of the particle. In some embodiments, a targeting compound is incorporated into the particle comprising a cationic polymer through a moiety incorporating the targeting compound into the particle comprising an anionic polymer. In some embodiments, the cationic polymer comprises one or more selected from the group consisting of cationic or polycationic peptides or proteins, including protamine, spermin or spermidine, poly-lysine, poly-arginine, cationic polysaccharides, including chitosan, cationic polymers, including poly(ethyleneimine), poly(propyleneimine), polybrene, polyallylamines, and polyvinylamine. In some embodiments, the polymer comprises a polyamidoamine (PAMAM) polymer.

[0744] Ill In some embodiments, a cationic polymer is a homopolymer selected from poly(ethylenimine), poly(propylenimine), polybrene, polyallylamine, polyvinylamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L-histidine, and poly(2-aminoethyl methacrylate), or a pharmaceutically acceptable salt thereof.

[0745] It is understood that polymers described herein can be linear or branched. In some embodiments, a cationic polymer is linear. In some embodiments, a cationic polymer is a linear polymer selected from poly(ethylenimine), poly(propylenimine), polybrene, polyallylamine, polyvinylamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L- histidine, and poly(2-aminoethyl methacrylate). In some embodiments, a cationic polymer is a branched polymer selected from poly(ethylenimine), poly(propylenimine), polybrene, polyallylamine, polyvinylamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L- histidine, and poly(2-aminoethyl methacrylate).

[0746] In some embodiments, the polymer comprises poly(ethyleneimine). In some embodiments, the poly(ethyleneimine) is a linear polymer. In some embodiments, the poly(ethyleneimine) is a branched polymer. In some embodiments, the poly(ethyleneimine) has a mean molar mass between 1000 Da and 150000 Da, between 5000 Da and 100000 Da, between 10000 Da and 50000 Da, between 15000 Da and 30000 Da, between 20000 Da and 25000 Da, or of about 22500 Da. In some embodiments, the poly(ethyleneimine) has a mean molar mass between 22500 Da and 150000 Da.

[0747] Particles described herein may also comprise polymers other than cationic polymers, i.e., noncationic polymers and / or anionic polymers. Collectively, anionic and neutral polymers are referred to herein as non-cationic polymers.

[0748] Particles comprising nucleic acid are also referred to as "polyplexes (PLX)" herein. Such PLX may comprise a lipid component, e.g., the lipid component of a targeting compound. Such particles containing polymer and lipid, e.g., functionalized lipid, are also referred to as lipidated polyplexes (LPLX). Polyamine derivatives (Viromers)

[0749] In some embodiments, the delivery vehicle comprises a polyamine derivative, e.g., a carboxylated polyamine derivative. Polyamines form polycations in solution, which facilitates the complex formation with polyanions such as nucleic acids.

[0750] In some embodiments, a polyamine derivative which is useful herein as delivery vehicle for polyanions comprises: a polyamine moiety comprising a plurality of amino groups; a plurality of carboxylated substituents comprising a carboxyl group bonded via a hydrophobic linker to amino groups of said polyamine moiety; and a plurality of hydrophobic substituents bonded to amino groups of said polyamine moiety.

[0751] In some embodiments, a polyamine derivative which is useful herein as delivery vehicle for polyanions comprises: a polyamine moiety comprising a plurality of amino groups; a plurality of carboxylated substituents comprising a carboxyl group bonded via a hydrophobic linker to amino groups of said polyamine moiety, wherein each of said carboxylated substituents comprises from 6 to 40 carbon atoms, preferably from 6 to 20 carbon atoms, and more preferably from 8 to 16 carbon atoms, and each of said hydrophobic linker may comprise from 1 to 3 heteroatoms selected from O, N, and S; and a plurality of hydrophobic substituents bonded to amino groups of said polyamine moiety, wherein each of said hydrophobic substituents comprises at least 2 carbon atoms, preferably from 6 to 40 carbon atoms, and may comprise from 1 to 3 heteroatoms selected from O, N, and S provided said hydrophobic substituent has at least 6 carbon atoms.

[0752] In some embodiments, each of said carboxylated substituents of said polyamine derivative comprises any one or more of the following moieties as said hydrophobic linker: alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, arylene, and combinations thereof; and / or each of said hydrophobic substituents of said polyamine derivative comprises any one or more of the following moieties: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, and combinations thereof. In some embodiments, a polyamine derivative which is useful herein as delivery vehicle for polyanions is a polyalkylenimine derivative having one or more carboxyalkyl substituents comprising from 6 to 40 carbon atoms, and one or more hydrophobic substituents selected from hydrocarbon substituents having at least 2 carbon atoms, preferably from 6 to 40 carbon atoms, wherein each of said hydrophobic substituents may be or may comprise an alkyl group and / or each of said hydrophobic substituents may be or may comprise an aryl group.

[0753] In some embodiments, the polyalkylenimine is selected from the group consisting of polyethylenimines, polypropylenimines, and polybutylenimines.

[0754] In some embodiments, the polyamine moiety of said polyamine derivative may comprise from 4 to 20000 nitrogen atoms, more preferably from 6 to 10000 nitrogen atoms, e.g., from 6 to 1000 nitrogen atoms, or from 6 to 100 nitrogen atoms per polyamine molecule.

[0755] In some embodiments, the polyamine moiety of said polyamine derivative may be a branched polyamine, preferably a branched polyalkylenimine.

[0756] In some embodiments, a carboxylated substitutent comprises one or two carboxyl groups, preferably one carboxyl group. In some embodiments, each carboxylated substitutent comprises from 6 to 40 carbon atoms, preferably from 6 to 20 carbon atoms, and more preferably from 8 to 16 carbon atoms. The hydrophobic linkers of said carboxylated substituents may comprise from 1 to 3, preferably, 1 or 2, heteroatoms selected from O, N, and S. Preferably, the heteroatoms are selected from O and S. In one embodiment, 1 or 2 heteroatoms selected from O, N and S, preferably O and S, may be contained in the hydrophobic linker. Thus, the carboxylated substituents may be carboxyhydrocarbyl groups, or they may be carboxyheterohydrocarbyl groups comprising from 1 to 3 heteroatoms selected from O, N, and S, preferably selected from O and S.

[0757] Among the plurality of carboxylated substituents of a molecule of said polyamine derivative, there may be exclusively carboxyhydrocarbyl groups, exclusively carboxyheterohydrocarbyl groups, or there may be carboxyhydrocarbyl groups and carboxyheterohydrocarbyl groups. In some embodiments, the plurality of carboxylated substituents are all carboxyhydrocarbyl groups. In some embodiments, the plurality of carboxylated substituents are all carboxyheterohydrocarbyl groups. Where the carboxylated substituents are carboxyhydrocarbyl groups, the hydrocarbyl moieties of said carboxyhydrocarbyl groups may be saturated aliphatic hydrocarbyl moieties, unsaturated aliphatic hydrocarbyl moieties, alicyclic hydrocarbyl moieties, aromatic hydrocarbyl moieties, or moieties comprising two or more moieties from the aforementioned list.

[0758] Examples of the 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. It is possible to replace 1 , 2 or 3, preferably 1 or 2, of the carbon atoms of the hydrocarbyl moieties of the carboxylated substituents by oxygen, nitrogen or sulfur, thereby forming carboxyheterohydrocarbyl moieties. It is understood that any such formal replacement by a heteroatom will include adjustment of bound hydrogen atoms to adjust to the valency of the exchanged heteroatom. In preferred embodiments, such carboxyheterohydrocarbyl moieties comprise one or more functional group selected from -0- , -S-, -N(H)C(O)-, -C(0)0- -OC(O)N(H)-, -C(0)-, -C(O)-N(H)-, -N(H)-C(O)-O-, -O-C(O)-, or -S- S- in the hydrophobic linker.

[0759] In some embodiments, the hydrophobic linkers are or comprise alkylene groups such as linear or branched alkylene groups, or the linkers are or comprise cycloalkylene groups. Alkylene groups may be n-alkylene or isoalkylene groups. Examples of alkylene groups are propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, dodecylene, tetradecylene or hexadecylene groups. Examples of cycloalkylene groups are cyclopentylene, cyclohexylene and cycloheptylene groups. Examples of alkylcycloalkyl groups are methylcyclopentylene, ethylcyclopentylene, propylcyclopentylene, butylcyclopentylene, pentylcyclopentylene, hexylcylopentylene, methylcyclohexylene, ethylcyclohexylene, propylcyclohexylene, butylcyclohexylene, pentylcyclohexylene and hexylcylohexylene. One or more of these may be combined in a hydrophobic linker.

[0760] In some embodiments, the carboxylated substituents are or comprise carboxyalkyl or carboxycycloalkyl groups comprising from 6 to 20 carbon atoms. Such carboxylated substituents may be selected from the group consisting of carboxy-n-alkyl groups, branched carboxyalkyl groups or cyclic carboxyalkyl groups and their constitution or conformation isomers. In a preferred embodiment, the carboxylalkyl groups are radicals of acids selected from hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, 2-cyclohexylacetic acid, 4- cyclohexylbutyric acid, 6-cyclohexylhexanoic acid, 2-(2', 3' or 4' ethylcyclohexyl)-acetic acid or 4-(2', 3' or 4' ethylcyclohexyl)-butyric acid or 6-(2', 3' or 4' ethylcyclohexyl)-hexanoic acid.

[0761] In some embodiments, the hydrophobic linkers are or comprise arylene groups and have from 6 to 20 carbon atoms. Aryl groups forming said arylene groups include aromatic hydrocarbyl groups (carbon-only aryl groups) and aromatic heterohydrocarbyl groups (heteroaryl groups). Examples of the former are phenyl, naphthyl, anthracenyl and phenanthryl. In some embodiments, nitrogen-containing heteroaryl groups have a pK value of <5 for avoiding additional cationic charges at neutral pH. Examples of such nitrogen-containing heteroaryl groups are indolyl groups pyrazinyl groups, pyridazinyl groups, pyrimidinyl groups, cinnolinyl groups, phthalazinyl groups and purinyl groups. In some embodiments, oxygen-containing heterohydrocarbyl groups that form hydroxy groups have a pK>12 for avoiding negative charges at neutral pH.

[0762] 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-isopropyl benzyl groups. Carboxyarylalkyl moieties may for example be radicals derived from from o, m or p- methyl benzoic acid, or o-, m- or p-ethyl benzoic acid. Carboxyalkylarylalkyl moieties may for example be o-, m- or p-methyl phenylacetic acid. Carboxyalkenylarylalkyl moieties may for example be or from o-, m- or p-methyl cinnamic acid.

[0763] Multiple carboxylated substituents such as those being or comprising carboxyalkyl groups present on the polyamine derivative may be the same or different. For simplicity, they may be the same. The carboxy group of the carboxylated substituent may be bound to any carbon atom of the hydrophobic linker. Preferably, the carboxy group is bound to a carbon atom as follows: if z is the number of carbon atoms in the longest carbon chain in the carboxylated substituent (such as the carboxyalkyl group) to the carbon atom that is bound to a polyamine nitrogen atom, the carboxy group is bound to a carbon atom at a position that is more than z / 2 atom positions away from the polyamine nitrogen, if the carbon atom bound to the polyamine nitrogen is counted as position 1. If the value of z / 2 is not an integer, the above definition leads to the position defined by the next integer > z / 2. In one embodiment, the carboxy group is bound to the carbon atom of the hydrophobic linker that is most remote (in terms of the number of carbon atoms) from the polyamine nitrogen atom to which the hydrophobic linker (alkylene chain in the case of carboxyalkyl groups) is connected. The carboxy group may be bound to the carbon atom that is farthest away from the polyamine nitrogen within the carboxylated substituent (or carboxyalkyl group), such as to the terminal (omega position) carbon atom of the carboxylated substituents (or carboxyalkyl group) in case of a linear carboxylated substituent.

[0764] In some embodiments, the hydrophobic substituents comprise from 2 to 40 carbon atoms, in some embodiments, from 3 to 40 carbon atoms, in some embodiments from 6 to 40 carbon atoms and in some embodiments from 6 to 20 carbon atoms. The hydrophobic substituents may comprise from 1 to 3, preferably 1 or 2, heteroatoms selected from 0, N, and S, provided said hydrophobic substituents comprise 6 or more carbon atoms. Preferably, the heteroatoms are selected from O and S. Thus, the hydrophobic substituents may be hydrocarbyl groups or heterohydrocarbyl groups, the latter comprising from 1 to 3 heteroatoms as mentioned before. Among the plurality of hydrophobic substituents of a molecule of said polyamine derivative, there may be exclusively hydrocarbyl groups, exclusively heterohydrocarbyl groups, or there may be hydrocarbyl groups and heterohydrocarbyl groups. In some embodiments, the plurality of hydrophobic substituents are all hydrocarbyl groups. In some embodiments, the plurality of hydrophobic substituents are all heterohydrocarbyl groups.

[0765] Where the hydrophobic substituents are hydrocarbyl groups, they may be selected from alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, cycloalkenyl groups, cycloalkylalkyl groups, alkylcycloalkyl groups, alkylcycloalkylalkyl groups, aryl groups, alkylaryl groups, arylalkyl groups, and alkylarylalkyl groups and groups comprising two or more groups from the aforementioned list. Provided the hydrophobic substituent comprises 6 or more carbon atoms, it is possible to replace 1, 2 or 3 of the carbon atoms of said hydrocarbyl groups by oxygen, nitrogen or sulfur, preferably oxygen or sulfur, thereby forming heterohydrocarbyl substituents. Such heterohydrocarbyl substituents may comprise functional groups selected from -0-, -S-, -N(H)C(O)-, -C(0)0-, -OC(O)N(H)-, -C(O)-, -C(0)- N(H)-, -N(H)-C(O)-O-, -O-C(O)-, or -S-S-.

[0766] In some embodiments, the hydrophobic substituents are or comprise alkyl groups such as linear or branched alkyl groups, or cycloalkyl groups. Alkyl groups may be n- alkyl or isoalkyl groups. Examples of alkyl groups are propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tetradecyl or hexadecyl groups. Examples of cycloalkyl groups are cyclopentyl, cyclohexyl and cycloheptyl groups.

[0767] 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 hexadecynyl groups.

[0768] Examples of cycloalkenyl groups are cyclopentenyl, cyclohexenyl and cycloheptenyl groups. Cycloalkylalkyl groups are groups wherein a cycloalkyl group is linked to an alkylene group corresponding to an alkyl group. Examples are cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl, cyclohexylethyl etc.

[0769] Alkylcycloalkyl groups are groups wherein an alkyl group is linked to a cycloalkylene group corresponding to a cycloalkyl group. Examples of alkylcycloalkyl groups are methylcyclopentyl, ethylcyclopentyl, propylcyclopentyl, butylcyclopentyl, pentylcyclopentyl, hexylcylopentyl, methylcyclohexyl, ethylcyclohexyl, propylcyclohexyl, butylcyclohexyl, pentylcyclohexyl and hexylcylohexyl.

[0770] Alkylcycloalkylalkyl groups are groups wherein an alkyl group is linked to a cycloalkylalkylene group.

[0771] In some embodiments, the hydrophobic substituent comprises an aryl group and has from 6 to 20, preferably from 7 to 15 carbon atoms. Aryl groups include aromatic hydrocarbyl groups (carbon-only aryl groups) and aromatic heterohydrocarbyl groups (heteroaryl groups). Examples of the former are phenyl, naphthyl and phenanthryl. In some embodiments, nitrogen-containing heteroaryl groups have a pK value of <5 for avoiding additional cationic charges at neutral pH. Examples of such nitrogen-containing heteroaryl groups are indolyl groups pyrazinyl groups, pyridazinyl groups, pyrimidinyl groups, cinnolinyl groups, phthalazinyl groups and purinyl groups. In some embodiments, oxygen-containing heterohydrocarbyl groups that form hydroxy groups have a pK>12 for avoiding negative charges at neutral pH.

[0772] 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.

[0773] Different hydrophobic substituents on a molecule of the polyamine derivative may be the same or may be different. For simplicity, they may be the same.

[0774] In some embodiments, the polyamine derivative has a linear polyethylenimine moiety of from 2 to 500 kDa (in terms of number average molecular weight), the carboxylated substituents have from 10 to 16 carbon atoms and are n-alkylcarboxylic acids and the hydrophobic substituents have from 1 to 12 carbon atoms and are alkyls, preferably n-alkyls, and / or alkylarylalkyls.

[0775] In some embodiments, the polyamine derivative has a branched polyethylenimine moiety of from 0.5 to 200 kDa (in terms of number average molecular weight), the carboxylated substituents have from 10 to 16 carbon atoms and are n-alkylcarboxylic acids and the hydrophobic substituents have from 1 to 12 carbon atoms and are alkyls, preferably n-alkyls, and / or alkylarylalkyls.

[0776] In some embodiments, the particle forming components comprise a compound comprising the following formula: Lipids

[0777] The terms "lipid" and "lipid-like material" are broadly defined herein as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also frequently denoted as amphiphiles. Lipids are usually insoluble or poorly soluble in water, but soluble in many organic solvents. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases. One of those phases consists of lipid bilayers, as they are present in vesicles, multilamellar / unilamellar liposomes, or membranes in an aqueous environment. Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). The hydrophilic groups may comprise polar and / or charged groups and include carbohydrates, phosphate, carboxylic, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other like groups.

[0778] As used herein, the term "hydrophobic" refers to any a molecule, moiety or group which is substantially immiscible or insoluble in aqueous solution. The term hydrophobic group includes hydrocarbons having at least 6 carbon atoms. The monovalent radical of a hydrocarbon is referred to as hydrocarbyl herein. The hydrophobic group can have functional groups (e.g., ether, ester, halide, etc.) and atoms other than carbon and hydrogen as long as the group satisfies the condition of being substantially immiscible or insoluble in aqueous solution.

[0779] The term "hydrocarbon" includes non-cyclic, e.g., linear (straight) or branched, hydrocarbyl groups, such as alkyl, alkenyl, or alkynyl as defined herein. It should be appreciated that one or more of the hydrogen atoms in alkyl, alkenyl, or alkynyl may be substituted with other atoms, e.g., halogen, oxygen or sulfur. Unless stated otherwise, hydrocarbon groups can also include a cyclic (alkyl, alkenyl or alkynyl) group or an aryl group, provided that the overall polarity of the hydrocarbon remains relatively nonpolar.

[0780] The term "alkyl" refers to a saturated linear or branched monovalent hydrocarbon moiety which may have one to thirty, typically one to twenty, often six to eighteen carbon atoms. Exemplary nonpolar alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, hexyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, and the like.

[0781] The term "alkenyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon double bond in which the total carbon atoms may be two to thirty, typically six to twenty often six to eighteen. Generally, the maximal number of carbon-carbon double bonds in the alkenyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenyl group by 2 and, if the number of carbon atoms in the alkenyl group is uneven, rounding the result of the division down to the next integer. 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 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds.

[0782] The term "alkynyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon triple bond in which the total carbon atoms may be two to thirty, typically six to twenty, often six to eighteen. Alkynyl groups can optionally have one or more carbon-carbon double bonds. Generally, the maximal number of carbon-carbon triple bonds in the alkynyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkynyl group by 2 and, if the number of carbon atoms in the alkynyl group is uneven, rounding the result of the division down to the next integer. For example, for an alkynyl group having 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, the alkynyl group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, more preferably 1 or 2 carbon-carbon triple bonds.

[0783] The term "alkylene" refers to a saturated linear or branched divalent hydrocarbon moiety which may have one to thirty, typically two to twenty, often four to twelve carbon atoms. Exemplary nonpolar alkylene groups include, but are not limited to, methylene, ethylene, trimethylene, hexamethylene, decamethylene, dodecamethylene, tetradecamethylene, hexadecamethylene, octadecmethylene, and the like.

[0784] The term "alkenylene" refers to a linear or branched divalent hydrocarbon moiety having at least one carbon-carbon double bond in which the total carbon atoms may be two to thirty, typically two to twenty, often four to twelve. Generally, the maximal number of carboncarbon double bonds in the alkenylene group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenylene group by 2 and, if the number of carbon atoms in the alkenylene group is uneven, rounding the result of the division down to the next integer. 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 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds.

[0785] The term "alkynylene" refers to a linear or branched divalent hydrocarbon moiety having at least one carbon-carbon triple bond in which the total carbon atoms may be two to thirty, typically two to twenty, often four to twelve. Alkynyl groups can optionally have one or more carbon carbon double bonds.

[0786] The term "cycloalkyl" represents cyclic non-aromatic versions of "alkyl" and "alkenyl" with preferably 3 to 14 carbon atoms, such as 3 to 12 or 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms (such as 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cylcodecyl, cylcodecenyl, and adamantyl. The cycloalkyl group may consist of one ring (monocyclic), two rings (bicyclic), or more than two rings (polycyclic).

[0787] The term "aryl" refers to a monoradical of an aromatic cyclic hydrocarbon. Preferably, the aryl group contains 3 to 14 (e.g., 5, 6, 7, 8, 9, or 10, such as 5, 6, or 10) carbon atoms which can be arranged in one ring (e.g., phenyl) or two or more condensed rings (e.g., naphthyl). Exemplary aryl groups include cyclopropenylium, cyclopentadienyl, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, and phenanthryl. 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 encompass fullerenes.

[0788] The term "aromatic" as used in the context of hydrocarbons means that the whole molecule has to be aromatic. For example, if a monocyclic aryl is hydrogenated (either partially or completely) the resulting hydrogenated cyclic structure is classified as cycloalkyl for the purposes of the present disclosure. Likewise, if a bi- or polycyclic aryl (such as naphthyl) is hydrogenated the resulting hydrogenated bi- or polycyclic structure (such as 1,2- dihydronaphthyl) is classified as cycloalkyl for the purposes of the present disclosure (even if one ring, such as in 1,2-dihydronaphthyl, is still aromatic).

[0789] As used herein, the term "amphiphilic" refers to a molecule having both a polar portion and a non-polar portion. Often, an amphiphilic compound has a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the nonpolar portion is insoluble in water. In addition, the polar portion may have either a formal positive charge, or a formal negative charge. Alternatively, the polar portion may have both a formal positive and a negative charge, and be a zwitterion or inner salt. For purposes of the disclosure, the amphiphilic compound can be, but is not limited to, one or a plurality of natural or non-natural lipids and lipid-like compounds.

[0790] The term "lipid-like material", "lipid-like compound" or "lipid-like molecule" relates to substances, in particular amphiphilic substances, that structurally and / or functionally relate to lipids but may not be considered as lipids in a strict sense. For example, the term includes compounds that are able to form amphiphilic layers as they are present in vesicles, multilamellar / unilamellar liposomes, or membranes in an aqueous environment and includes surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties. Generally speaking, the term includes molecules, which comprise hydrophilic and hydrophobic moieties with different structural organization, which may or may not be similar to that of lipids. Examples of lipid-like compounds capable of spontaneous integration into cell membranes include functional lipid constructs such as synthetic function-spacer-lipid constructs (FSL), synthetic function-spacer-sterol constructs (FSS) as well as artificial amphipathic molecules. Lipids comprising two long alkyl chains and a polar head group are generally cylindrical. The area occupied by the two alkyl chains is similar to the area occupied by the polar head group. Such lipids have low solubility as monomers and tend to aggregate into planar bilayers that are water insoluble. Traditional surfactant monomers comprising only one linear alkyl chain and a hydrophilic head group are generally cone shaped. The hydrophilic head group tends to occupy more molecular space than the linear alkyl chain. In some embodiments, surfactants tend to aggregate into spherical or elliptoid micelles that are water soluble. While lipids also have the same general structure as surfactants - a polar hydrophilic head group and a nonpolar hydrophobic tail - lipids differ from surfactants in the shape of the monomers, in the type of aggregates formed in solution, and in the concentration range required for aggregation. As used herein, the term "lipid" is to be construed to cover both lipids and lipid-like materials unless otherwise indicated herein or clearly contradicted by context.

[0791] Generally, lipids may be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from condensation of ketoacyl subunits), sterol lipids and prenol lipids (derived from condensation of isoprene subunits). Although the term "lipid" is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as steroids, i.e., sterol-containing metabolites such as cholesterol or a derivative thereof. 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.

[0792] Fatty acids, or fatty acid residues are a diverse group of molecules made of a hydrocarbon chain that terminates with a carboxylic acid group; this arrangement confers the molecule with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in water. The carbon chain, typically between four and 24 carbons long, may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains a double bond, there is the possibility of either a cis or trans geometric isomerism, which significantly affects the molecule's configuration. Cis-double bonds cause the fatty acid chain to bend, an effect that is compounded with more cis double bonds in the chain. Other major lipid classes in the fatty acid category are the fatty esters and fatty amides. Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the best-known being the fatty acid triesters of glycerol, called triglycerides. The word "triacylglycerol" is sometimes used synonymously with "triglyceride". In these compounds, the three hydroxyl groups of glycerol are each esterified, typically by different fatty acids. Additional subclasses of glycerolipids are represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic linkage. The glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to two fatty acid-derived "tails" by ester linkages and to one "head" group by a phosphate ester linkage. Examples of glycerophospholipids, usually referred to as phospholipids (though sphingomyelins are also classified as phospholipids) are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer).

[0793] Sphingolipids are a complex family of compounds that share a common structural feature, a sphingoid base backbone. The major sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with an amide-linked fatty acid. The fatty acids are typically saturated or monounsaturated with chain lengths from 16 to 26 carbon atoms. The major phosphosphingolipids of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and fungi have phytoceramide phosphoinositols and mannose-containing headgroups. The glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base. Examples of these are the simple and complex glycosphingolipids such as cerebrosides and gangliosides.

[0794] Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, are an important component of membrane lipids, along with the glycerophospholipids and sphingomyelins.

[0795] Saccharolipids describe compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers. In the saccharolipids, a monosaccharide substitutes for the glycerol backbone present in glycerolipids and glycerophospholipids. The most familiar saccharolipids are the acylated glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gram-negative bacteria. Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty-acyl chains. The minimal lipopolysaccharide required for growth in E. coli is Kdo2-Lipid A, a hexa-acylated disaccharide of glucosamine that is glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues. Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

[0796] According to the disclosure, lipids and lipid-like materials may be cationic, anionic or neutral. Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH.

[0797] Cationic / Cationically ionizable lipids

[0798] In some embodiments, the particles described herein comprise at least one cationic or cationically ionizable lipid as particle forming agent. Cationic or cationically ionizable lipids contemplated for use herein include any cationic or cationically ionizable lipids (including lipid- like materials) which are able to electrostatically bind nucleic acid. In some embodiments, cationic or cationically ionizable lipids contemplated for use herein can be associated with nucleic acid, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.

[0799] As used herein, a "cationic lipid" refers to a lipid or lipid-like material having a net positive charge. Generally, cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries the positive charge. In some embodiments, a cationic lipid has a net positive charge only at certain pH, in particular acidic pH, while it has preferably no net positive charge, preferably has no charge, i.e., it is neutral, at a different, preferably higher pH such as physiological pH. This ionizable behavior is thought to enhance efficacy through helping with endosomal escape and reducing toxicity as compared with particles that remain cationic at physiological pH.

[0800] As used herein, a "cationically ionizable lipid" refers to a lipid or lipid-like material which has a net positive charge or is neutral, i.e., which is not permanently cationic. Thus, depending on the pH of the composition in which the cationically ionizable lipid is solved, the cationically ionizable lipid is either positively charged or neutral. For purposes of the present disclosure, cat ionical ly ionizable lipids are covered by the term "cationic lipid" unless contradicted by the circumstances.

[0801] In some embodiments, the cationic or cationically ionizable lipid comprises a head group which includes at least one nitrogen atom (N) which is positive charged or capable of being protonated, e.g., under physiological conditions.

[0802] Examples of cationic or cationically ionizable lipids include, but are not limited to N,N- dimethyl-2,3-dioleyloxypropylamine (DODMA), l,2-dioleoyl-3-trimethylammonium propane (DOTAP); l,2-di-0-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N— (N',N'- dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); l,2-dioleoyl-3-dimethylammonium-propane (DODAP); l,2-diacyloxy-3- dimethylammonium propanes; l,2-dialkyloxy-3-dimethylammonium propanes; dioctadecyldimethyl ammonium chloride (DODAC), l,2-distearyloxy-N,N-dimethyl-3- aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2-dimyristoyl-3- trimethylammonium propane (DMTAP), l,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), and 2,3-dioleoyloxy- N-[2(spermine carboxamide)ethyl]-N,N- dimethyl-l-propanamium trifluoroacetate (DOSPA), l,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3- beta-oxybutan-4-oxy)-l-(cis,cis-9,12-oc-tadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5- en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-l-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), l,2-N,N'-dioleylcarbamyl-3- dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), l,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-

[0803] Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl- [l,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin- MC3-DMA), N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-l- propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3- bis(dodecyloxy)-l-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N- dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (GAP-DMRIE), N-(2-Aminoethyl)- N,N-dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (£AE-DMRIE), N-(4- carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-l-aminium (DOBAQ), 2-({8-[(3(3)- cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l- yloxy]propan-l-amine (Octyl-CLinDMA), l,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), l,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), Nl-[2-((lS)-l-[(3- aminopropyl)arnino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]- benzamide (MVL5), l,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3- bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropan-l-amonium bromide (DLRIE), N-(2- aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-l-aminium bromide (DMORIE), di((Z)-non-2-en-l-yl) 8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-l-amine (DLDMA), N,N-dimethyl-2,3- bis(tetradecyloxy)propan-l-amine (DMDMA), Di((Z)-non-2-en-l-yl)-9-((4-

[0804] (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 98N12-5), 1- [2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2 hydroxydodecyl)amino]ethyl]piperazin- l-yl]ethyl]amino]dodecan-2-ol (lipidoid C12-200).

[0805] In some embodiments, the cationic or cationically ionizable lipid is DOTMA. In some embodiments, the cationic or cationically ionizable lipid is DODMA.

[0806] DOTMA is a cationic lipid with a quaternary amine headgroup. The structure of DOTMA may be represented as follows:

[0807] DODM A is an ionizable cationic lipid with a tertiary amine headgroup. The structure of DODMA may be represented as follows:

[0808] In some embodiments, the cationic or cationically ionizable lipid may comprise from about 10 mol % to about 95 mol %, from about 20 mol % to about 95 mol %, from about 20 mol % to about 90 mol %, from about 30 mol % to about 90 mol %, from about 40 mol % to about 90 mol %, or from about 40 mol % to about 80 mol % of the total lipid present in the particle.

[0809] Additional lipids

[0810] The particles described herein may also comprise lipids (including lipid-like materials) other than cationic or cationically ionizable lipids (also collectively referred to herein as cationic lipids), i.e., non-cationic lipids (including non-cationic or non-cationically ionizable lipids or lipid-like materials). Collectively, anionic and neutral lipids or lipid-like materials are referred to herein as non-cationic lipids. Optimizing the formulation of nucleic acid particles by addition of other hydrophobic moieties, such as cholesterol and lipids, in addition to a cationic or cationically ionizable lipid may enhance particle stability and efficacy of nucleic acid delivery. One or more additional lipids may or may not affect the overall charge of the nucleic acid particles. In some embodiments, the one or more additional lipids are a non-cationic lipid or lipid-like material. The non-cationic lipid may comprise, e.g., one or more anionic lipids and / or neutral lipids. As used herein, an "anionic lipid" refers to any lipid that is negatively charged at a selected pH. As used herein, a "neutral lipid" refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH.

[0811] In some embodiments, the nucleic acid particles described herein comprise a cationic or cationically ionizable lipid and one or more additional lipids.

[0812] Without wishing to be bound by theory, the amount of the cationic or cationically ionizable lipid compared to the amount of the one or more additional lipids may affect important nucleic acid particle characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the nucleic acid. Accordingly, in some embodiments, the molar ratio of the cationic or cationically ionizable lipid to the one or more additional lipids is from about 10:0 to about 1:9, about 4:1 to about 1:2, about 4:1 to about 1:1, about 3:1 to about 1:1, or about 3:1 to about 2:1. In some embodiments, the one or more additional lipids comprised in the nucleic acid particles described herein comprise one or more of the following: neutral lipids, steroids, and combinations thereof.

[0813] In some embodiments, the one or more additional lipids comprise a neutral lipid which is a phospholipid. In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins. Specific phospholipids that can be used include, but are not limited to, phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin. Such phospholipids include in particular diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), l-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 (DPyPE), l,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPG), 1,2-dipalmitoyl-sn- glycero-3-phospho-(l'-rac-glycerol) (DPPG), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM), and further 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.

[0814] 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. Thus, in some embodiments, the nucleic acid particles described herein comprise (1) a cationic or cationically ionizable lipid, and a phospholipid such as DSPC or DOPE or (2) a cationic or cationically ionizable lipid and a phospholipid such as DSPC or DOPE and cholesterol.

[0815] 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.

[0816] DSPC is a neutral phospholipid. The structure of DSPC may be represented as follows:

[0817] DOPE is a neutral phospholipid. The structure of DOPE may be represented as follows:

[0818] The structure of cholesterol may be represented as follows: In some embodiments, nucleic acid particles described herein do not include a polymer conjugated lipid such as a pegylated lipid.

[0819] In some embodiments, the additional lipid (e.g., one or more phospholipids and / or cholesterol) may comprise from about 0 mol % to about 90 mol %, from about 0 mol % to about 80 mol %, from about 2 mol % to about 80 mol %, from about 5 mol % to about 80 mol %, from about 5 mol % to about 60 mol %, from about 5 mol % to about 50 mol %, from about 7.5 mol % to about 50 mol %, or from about 10 mol % to about 40 mol % of the total lipid present in the particle. In some embodiments, the additional lipid (e.g., one or more phospholipids and / or cholesterol) comprises about 10 mol %, about 15 mol %, or about 20 mol % of the total lipid present in the particle.

[0820] In some embodiments, the additional lipid comprises a mixture of: (i) a phospholipid such as DOPE; and (ii) cholesterol or a derivative thereof. In some embodiments, the molar ratio of the phospholipid such as DOPE to the cholesterol or a derivative thereof is from about 9:0 to about 1:10, about 2:1 to about 1:4, about 1:1 to about 1:4, or about 1:1 to about 1:3.

[0821] Polymer-conjugated lipids

[0822] In some embodiments, the particles described herein may comprise at least one polymer- conjugated lipid. In some embodiments, the polymer-conjugated lipid comprises an amphiphilic derivative of a polymer which is part of a targeting compound and / or a polymer- conjugated lipid which is not part of a targeting compound. A polymer-conjugated lipid is typically a molecule comprising a lipid portion and a polymer portion conjugated thereto. In some embodiments, the polymer of the polymer-conjugated lipid is a polymer as described herein for the targeting compound. In some embodiments, a polymer-conjugated lipid is a PEG-conjugated lipid, also referred to herein as 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, a polymer-conjugated lipid is a polysarcosine-conjugated lipid, also referred to herein as sarcosinylated lipid or pSar- lipid. The term "sarcosinylated lipid" refers to a molecule comprising both a lipid portion and a polysarcosine portion. In some embodiments, a polymer-conjugated lipid is designed to sterically stabilize a lipid particle by forming a protective hydrophilic layer that shields the hydrophobic lipid layer. In some embodiments, a polymer-conjugated lipid can reduce its association with serum proteins and / or the resulting uptake by the reticuloendothelial system when such lipid particles are administered in vivo.

[0823] Polyethyleneglycol (PEG)-conjugated lipids

[0824] In some embodiments, the particles described herein comprise a PEG-conjugated lipid.

[0825] In some embodiments, the PEG-conjugated lipid (pegylated lipid) is a lipid having the structure of the following general formula: or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: each of R12and R13is each independently a straight or branched, alkyl or alkenyl chain containing from 10 to 30 carbon atoms, wherein the alkyl / alkenyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.

[0826] In some embodiments of this formula, each of R12and R13is independently a straight alkyl chain containing from 10 to 18 carbon atoms, preferably from 12 to 16 carbon atoms.

[0827] In some embodiments of this formula, R12and R13are identical. In some embodiments, each of R12and R13is a straight alkyl chain containing 12 carbon atoms. In some embodiments, each of R12and R13is a straight alkyl chain containing 14 carbon atoms. In some embodiments, each of R12and R13is a straight alkyl chain containing 16 carbon atoms.

[0828] In some embodiments of this formula, R12and R13are different. In some embodiments, one of R12and R13is a straight alkyl chain containing 12 carbon atoms and the other of R12and R13is a straight alkyl chain containing 14 carbon atoms.

[0829] In some embodiments of this formula, w has a mean value ranging from 40 to 50, such as a mean value of 45.

[0830] 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 from about 400 to about 6000 g / mol, such as from about 1000 to about 5000 g / mol, from about 1500 to about 4000 g / mol, or from about 2000 to about 3000 g / mol. In some embodiments, each of R12and R13is a straight alkyl chain containing 14 carbon atoms and w has a mean value of 45.

[0831] Various PEG-conjugated lipids are known in the art and include, but are not limited to pegylated diacylglycerol (PEG-DAG) such as l-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2' ,3 '-di(tetradecanoyloxy)propyl-l-O-(®- methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as ®-methoxy(polyethoxy)ethyl-N-(2,3- di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(® methoxy(polyethoxy)ethyl)carbamate, and the like.

[0832] 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:

[0833] In some embodiments, the PEG-conjugated lipid (pegylated lipid) is DMG-PEG 2000, e.g., having the following structure:

[0834] In some embodiments, the PEG-conjugated lipid (pegylated lipid) has the following structure: wherein n has a mean value ranging from 30 to 60, such as about 50. In some embodiments, the PEG-conjugated lipid (pegylated lipid) is PEG2000-C-DMA which preferably refers to 3-N- [(w-methoxy polyethylene glycol)2000)carbamoyl]-l,2-dimyristyloxy-propylamine (MPEG-(2 kDa)-C-DMA) or methoxy-polyethylene glycol-2,3-bis(tetradecyloxy)propylcarbamate (2000). In some embodiments, nucleic acid particles described herein may comprise one or more PEG- conjugated lipids or pegylated lipids as described in WO 2017 / 075531 and WO 2018 / 081480, the entire contents of each of which are incorporated herein by reference for the purposes described herein.

[0835] In some embodiments, the pegylated lipid comprises from about 1 mol % to about 10 mol %, preferably from about 1 mol % to about 5 mol %, more preferably from about 1 mol % to about 2.5 mol % of the total lipid present in the nucleic acid compositions / formulations and nucleic acid particles described herein.

[0836] Embodiments of Lipoplex Particles

[0837] In some embodiments of the present disclosure, the RNA described herein may be present in lipoplex particles.

[0838] Lipoplexes ( LPX) are electrostatic complexes which are generally formed by mixing preformed cationic lipid liposomes with anionic nucleic acid. Formed lipoplexes possess distinct internal arrangements of molecules that arise due to the transformation from liposomal structure into compact nucleic acid lipoplexes.

[0839] In some embodiments, liposomes are self-closed unilamellar or multilamellar vesicular particles wherein the lamellae comprise lipid bilayers and the encapsulated lumen comprises an aqueous phase. A prerequisite for using liposomes for nanoparticle formation is that the lipids in the mixture as required are able to form lamellar (bilayer) phases in the applied aqueous environment.

[0840] In certain embodiments, the nucleic acid lipoplex particles include both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

[0841] In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In specific embodiments, the molar ratio may be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2:1. Nucleic acid lipoplex particles described herein have an average diameter that in some embodiments ranges from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about 500 nm, or from about 350 nm to about 400 nm. In specific embodiments, the nucleic acid lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 675 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm. In some embodiments, the nucleic acid lipoplex particles have an average diameter that ranges from about 250 nm to about 700 nm. In some embodiments, the nucleic acid lipoplex particles have an average diameter that ranges from about 300 nm to about 500 nm. In an exemplary embodiment, the nucleic acid lipoplex particles have an average diameter of about

[0842] 400 nm.

[0843] Embodiments of Lipid nanoparticles (LNPs)

[0844] In some embodiments, RNA described herein is present in the form of lipid nanoparticles (LNPs). The LNP may comprise any lipid capable of forming a particle to which nucleic acid molecules are attached, or in which nucleic acid molecules are encapsulated.

[0845] In general, lipid nanoparticles are obtainable from direct mixing of nucleic acid, e.g., RNA, in an aqueous phase with lipids in a phase comprising an organic solvent, such as ethanol. In that case, lipids or lipid mixtures can be used for particle formation, which do not form lamellar (bilayer) phases in water.

[0846] LNPs typically comprise four components: cationically ionizable lipid, neutral lipids such as phospholipids, a steroid such as cholesterol, and a polymer-conjugated lipid such as PEG-lipid. LNPs may be prepared by mixing lipids dissolved in ethanol with nucleic acid in an aqueous buffer.

[0847] In some embodiments, the LNP comprises from 40 to 60 mol percent, 40 to 55 mol percent, from 45 to 55 mol percent, or from 45 to 50 mol percent of the cationically ionizable lipid. In some embodiments, the neutral lipid is present in a concentration ranging from 5 to 15 mol percent, from 7 to 13 mol percent, or from 9 to 11 mol percent.

[0848] In some embodiments, the steroid is present in a concentration ranging from 30 to 50 mol percent, from 30 to 45 mol percent, from 35 to 45 mol percent or from 35 to 43 mol percent. In some embodiments, the LNP comprises from 1 to 10 mol percent, from 1 to 5 mol percent, or from I to 2.5 mol percent of the polymer-conjugated lipid.

[0849] In some embodiments, the LNP comprises from 45 to 55 mol percent of a cationically ionizable lipid; from 5 to 15 mol percent of a neutral lipid; from 30 to 45 mol percent of a steroid; from 1 to 5 mol percent of a polymer-conjugated lipid; and the nucleic acid, encapsulated within or associated with the lipid nanoparticle.

[0850] In some embodiments, the mol percent is determined based on total mol of lipid present in the lipid nanoparticle. In some embodiments, the mol percent is determined based on total mol of cationically ionizable lipid, neutral lipid, steroid and polymer-conjugated lipid present in the lipid nanoparticle.

[0851] 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.

[0852] In some embodiments, the steroid is cholesterol.

[0853] In some embodiments, the polymer conjugated lipid is a pegylated lipid, e.g., a pegylated lipid as described above.

[0854] In some embodiments, the cationically ionizable lipid component of the LNPs has the structure of Formula (III):

[0855] (HI) or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: one of L1or L2is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-, and the other of L1or L2is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O- or a direct bond;

[0856] G1and G2are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene;

[0857] G3is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;

[0858] Rais H or C1-C12 alkyl;

[0859] R1and R2are each independently C6-C24 alkyl or C6-C24 alkenyl;

[0860] R3is H, OR5, CN, -C(=O)OR4, -OC(=O)R4or -NR5C(=O)R4;

[0861] R4is C1-C12 alkyl;

[0862] R5is H or C1-C6 alkyl; and x is 0, 1 or 2.

[0863] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIA) or (IIIB):

[0864] (IIIA) (IIIB) wherein:

[0865] A is a 3 to 8-membered cycloalkyl or cycloalkylene ring;

[0866] R6is, at each occurrence, independently H, OH or C1-C24 alkyl; n is an integer ranging from 1 to 15.

[0867] In some of the foregoing embodiments of Formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[0868] In other embodiments of Formula (III), the lipid has one of the following structures (IIIC) or (HID):

[0869] (IIIC) (HID) wherein y and z are each independently integers ranging from 1 to 12. In any of the foregoing embodiments of Formula (III), one of L1or L2is -O(C=O)-. For example, in some embodiments each of L1and L2are -O(C=O)-. In some different embodiments of any of the foregoing, L1and L2are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments each of L1and L2is -(C=O)O-.

[0870] In some different embodiments of Formula (III), the lipid has one of the following structures (IIIE) or (IIIF): (IIIE) (IIIF)

[0871] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIG), (IIIH), (Illi), or (IIIJ):

[0872] (IIII) (IIIJ)

[0873] In some of the foregoing embodiments of Formula (III), n is an integer ranging from 2 to 12, for example 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.

[0874] In some other 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. In some of the foregoing embodiments of Formula (III), R6is H. In other of the foregoing embodiments, R6is C1-C24 alkyl. In other embodiments, R6is OH.

[0875] In some embodiments of Formula (III), G3is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G3is linear C1-C24 alkylene or linear C1-C24 alkenylene.

[0876] In some other foregoing embodiments of Formula (III), R1or R2, or both, is C6-C24 alkenyl. For example, in some embodiments, R1and R2each, independently have the following structure: wherein:

[0877] R7aand R7bare, at each occurrence, independently H or C1-C12 alkyl; and a is an integer from 2 to 12, wherein R7a, R7band a are each selected such that R1and R2each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12.

[0878] In some of the foregoing embodiments of Formula (III), at least one occurrence of R7ais H. For example, in some embod...

Claims

Claims1. A particle comprising:(a) one or more particle forming components, and(b) a targeting compound comprising:(i) a moiety incorporating the targeting compound into the particle, and(ii) a moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag, wherein the particle carries RNA encoding a polypeptide comprising a cytokine or a functional variant thereof.

2. The particle of claim 1, wherein the targeting compound comprises the formula:L-X1-P-X2-B whereinP is absent or comprises a polymer,L comprises a moiety incorporating the targeting compound into the particle,B comprises a moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag,XI is absent or a first linking moiety, andX2 is absent or a second linking moiety.

3. The particle of claim 1 or 2, wherein the particle comprises a lipid particle, a polymer particle, or a mixture thereof.

4. The particle of any one of claims 1 to 3, wherein the particle comprises a lipid particle.

5. The particle of any one of claims 1 to 4, wherein the moiety incorporating the targeting compound into the particle comprises a hydrophobic moiety6. The particle of any one of claims 1 to 5, wherein the moiety incorporating the targeting compound into the particle comprises a moiety selected from vitamin E, dialkylamine, diacylglyceride and ceramide.

7. The particle of any one of claims 1 to 6, wherein the moiety incorporating the targeting compound into the particle comprises two C8-C24 hydrocarbon chains.

8. The particle of any one of claims 1 to 7, wherein the moiety incorporating the targeting compound into the particle comprises a lipid.

9. The particle of any one of claims 1 to 8, wherein the moiety incorporating the targeting compound into the particle comprises a phospholipid.10 The particle of any one of claims 1 to 9, wherein the moiety incorporating the targeting compound into the particle comprises a moiety selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine), and mixtures thereof.

11. The particle of any one of claims 2 to 10, wherein P comprises a hydrophilic polymer.

12. The particle of any one of claims 2 to 11, wherein P is selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), derivatives and combinations thereof.

13. The particle of any one of claims 2 to 12, wherein X2 comprises the reaction product of a thiol or cysteine reactive group with a thiol or cysteine group of a compound comprising the moiety B.

14. The particle of claim 13, wherein the thiol or cysteine reactive group comprises a maleimide group.

15. The particle of any one of claims 2 to 14, wherein the targeting compound comprises the reaction product of l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)] with a compound comprising the formula SH(CH2)nC(O)-B, wherein n ranges from 1 to 5 and preferably n is 2.

16. The particle of any one of claims 1 to 15, wherein the targeting compound comprises a compound of the formula:

17. The particle of any one of claims 1 to 16, wherein the moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag comprises a moiety binding to a cell surface antigen on immune cells.

18. The particle of any one of claims 1 to 16, wherein the moiety selected from the group consisting of a moiety binding to a cell surface antigen on immune cells, a tag, and a moiety binding to a tag comprises a moiety selected from the group consisting of a tag and a moiety binding to a tag and the particle further comprises a docking compound comprising:(i) a moiety binding to the moiety selected from the group consisting of a tag and a moiety binding to a tag, and(ii) a moiety binding to a cell surface antigen on immune cells.

19. The particle of claim 18, wherein the docking compound comprises the formula:B'-X3-B" whereinB' comprises a moiety binding to the moiety selected from the group consisting of a tag and a moiety binding to a tag,X3 is absent or a linking moiety, andB" comprises a moiety binding to a cell surface antigen on immune cells.

20. The particle of claim 18 or 19, wherein the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a tag and the moiety binding to the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a moiety binding to the tag.

21. The particle of claim 18 or 19, wherein the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a moiety binding to a tag and the moiety binding to the moiety selected from the group consisting of a tag and a moiety binding to a tag comprises a tag to which the moiety binding to a tag binds.

22. The particle of any one of claims 1 to 21, wherein the moiety binding to a cell surface antigen on immune cells comprises a peptide or polypeptide.

23. The particle of any one of claims 1 to 22, wherein the moiety binding to a cell surface antigen on immune cells comprises an antibody or antibody-like molecule.

24. The particle of claim 23, wherein the antibody-like molecule comprises an antibody fragment or DARPin.

25. The particle of any one of claims 1 to 24, wherein the immune cells comprise immune effector cells.

26. The particle of any one of claims 1 to 25, wherein the immune cells comprises T cells.

27. The particle of any one of claims 1 to 26, wherein the immune cells comprise CD8+ and / or CD4+ T cells.

28. The particle of any one of claims 1 to 27, wherein the cell surface antigen on immune cells is characteristic for the immune cells.

29. The particle of any one of claims 1 to 28, wherein the cell surface antigen on immune cells is selected from the group consisting of CD4, CD8 and CD3.

30. The particle of any one of claims 1 to 16 and 18 to 29, wherein the moiety binding to a tag comprises a peptide or polypeptide.

31. The particle of any one of claims 1 to 16 and 18 to 30, wherein the moiety binding to a tag comprises an antibody or antibody-like molecule.

32. The particle of claim 31, wherein the antibody-like molecule comprises an antibody fragment or DARPin.

33. The particle of any one of claims 1 to 16 and 18 to 32, wherein the tag comprises a peptide or polypeptide.

34. The particle of any one of claims 1 to 16 and 18 to 33, wherein the tag comprises a peptide tag.

35. The particle of any one of claims 1 to 16 and 18 to 34, wherein the tag comprises an ALFA-tag.

36. The particle of any one of claims 1 to 16 and 18 to 35, wherein the tag comprises an ALFA-tag and the moiety binding to the tag comprises a VHH domain comprising the CDR1 sequence VTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY.

37. The particle of any one of claims 1 to 36, wherein the particle is a non-viral particle.

38. The particle of any one of claims 1 to 37, wherein the particle is a nanoparticle.

39. The particle of any one of claims 1 to 38, wherein the particle is a lipid nanoparticle (LNP).

40. The particle of any one of claims 1 to 39, wherein the cytokine comprises an interleukin.

41. The particle of any one of claims 1 to 40, wherein the cytokine comprises interleukin 2.

42. A method for delivering a polypeptide comprising a cytokine or a functional variant thereof to immune cells expressing a cell surface antigen, comprising adding to the immune cells a composition comprising particles of any one of claims 1 to 41, wherein the moiety binding to a cell surface antigen on immune cells binds to the cell surface antigen expressed by the immune cells.

43. The method of claim 42, which is a method for immunomodulation of immune cells.

44. The method of claim 42 or 43, which is a method for inducing proliferation of immune cells.

45. A method for inducing proliferation of immune cells, comprising adding to the immune cells a composition comprising particles of any one of claims 1 to 41, wherein the moiety binding to a cell surface antigen on immune cells binds to a cell surface antigen expressed by the immune cells.

46. The method of any one of claim 42 to 45, wherein the immune cells are present ex vivo or in vitro.

47. The method of any one of claim 42 to 45, wherein the immune cells are present in a subject and the method comprises administering the composition to the subject.

48. A method for treating a subject comprising:(i) preparing ex vivo immune cells using the method of any one of claims 42 to 46, and(ii) administering the immune cells to the subject.

49. A method for treating a subject comprising administering to the subject a composition comprising particles of any one of claims 1 to 41.

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